Immunoisolation device

ABSTRACT

Provided herein is technology relating to immunoisolation of cells and tissues, including, but not exclusively, to compositions, methods, and kits for encapsulating cells and/or tissues within an immunoisolating device to protect the cells/or tissues from host immune rejection.

This application is a continuation of U.S. patent application Ser. No.15/755,242, filed Feb. 26, 2018, which is a 371 U.S. National PhaseEntry of International Application No. PCT/US2016/048673, filed Aug. 25,2016, which claims priority to U.S. provisional patent application Ser.No. 62/211,175, filed Aug. 28, 2015, each of which is incorporatedherein by reference in its entirety.

FIELD

Provided herein is technology relating to immunoisolation of cells andtissues, including, but not exclusively, to compositions, methods, andkits for encapsulating cells and/or tissues within an immunoisolatingdevice to protect the cells/or tissues from host immune rejection.

BACKGROUND

Premature ovarian failure (POF) is a common consequence of cytotoxictreatments, e.g., used to treat cancer and autoimmune disease, due toextreme ovarian toxicity of chemotherapy and bone marrow transplantation(see, e.g., Darlington et al. (1999) Am J Epidemiol 150(3): 245-54;Cohen et al. (2011) J Pediatr Adolesc Gynecol 24(2): 101-03; Brauner etal. (1998) Bone Marrow Transplant 21(3): 287-90; Wallace et al. (2000)Hosp Med 61(8): 550-57; Woodruff et al. (2007) Cancer Treat Res 138:201-17). Modern cancer therapy has improved the survival rate to over80% for children and young adults diagnosed with cancer in the UnitedStates, and now these cancer survivors face long-term health problems(see, e.g., Ness at al. (2015) J Clin Oncol 33(5): 479-91; Feuer et al.(2009) Cancer Epidemiol Biomarkers Prev 18(4): 1033-40; Mariotta et al.(2015) Cancer Epidemiol Biomarkers Prev 24(4): 653-63; Hudson et al.(2014) 14(1): 61-70). In female cancer survivors, POF causes sterilityand several problems associated with a loss of ovarian endocrinefunction: premature osteopenia, muscle wasting, and acceleratedcardiovascular diseases. These long lasting effects are significant,particularly for young girls reaching puberty. Extant treatment optionsfor POF rely on hormone replacement therapy (HRT), which deliversunregulated, non-physiological levels of estrogen that interfere withgrowth in peripubertal girls and predisposes recipients to cancer andthrombotic events. However, although these therapies have beenextensively studied in menopausal adult women, long-term safety data inchildren is scant. The risk of synthetic hormones is attributed tounnaturally elevated levels following their administration, as opposedto the normal fluctuating physiologic levels maintained by the finelytuned feedback mechanism provided by healthy ovaries. Further, noalternative form of therapy for POF is available to young girls.

SUMMARY

In some embodiments, provided herein is a technology related totransplant of cells. For example, in some embodiments the technologyfinds use in the transplant of cells that produce a bioactive moleculeinto a patient in need of the bioactive molecule. In some embodiments,the technology relates to the transplant of ovarian cells to a femalecancer patient. In some embodiments, the technology relates to treatmentof menopause and other conditions. That is, in some embodiments, thetechnology relates to a donor ovarian transplant strategy that providesdelivery of natural estrogen at physiologic levels, while simultaneouslyreestablishing hormonal feedback regulation. The technology provides abioengineered matrix that supports follicle survival and function,which, in an immune privileged environment, prevents rejection by therecipient while continuing to produce estrogen and progesterone underconditions similar to normal physiologic regulation. In someembodiments, the technology finds use in treating young girls who areforced to endure ovarian failure as the result of cancer treatmentstrategies. The technology provides for avoiding the deleterious effectsof estrogen and progesterone deficiency and risks associated withsynthetic hormonal replacement therapy, thus promoting normaldevelopment and puberty, and an otherwise healthy life.

The technology provides a regenerative therapy employing aspects ofengineering, materials, chemistry, and life sciences to create syntheticconstructs to direct tissue regeneration and restoration of biologicalfunction. For example, in some embodiments synthetic hydrogels find use,which provide a three-dimensional environment similar to theextracellular matrix (ECM), allow diffusion of nutrients, and can bemodified to present many biological functions, e.g., matrix-to-celladhesion and biodegradation. In some embodiments, fibrin hydrogels finduse for transplantation of ovarian tissue or cells, or fortransplantation of isolated human follicles. Fibrin gels have anintrinsic bioactivity and provide a physical connectivity for thefollicles in a graft and between the graft and the device, both of whichcontribute to tissue survival. Furthermore, poly(ethyleneglycol) (PEG)is a synthetic multifunctional hydrophilic polymer that is extensivelyused in biomedical applications and tissue engineering. PEG-basedhydrogels are not immunogenic and have biocompatible chemistry withinphysiological conditions. The baseline biological inertness ofbiomaterials such as these provides for incorporating custom designedbiological moieties that are naturally found in the extra cellularmatrix (ECM). For example, in some embodiments integrin bindingmolecules derived from ECM proteins, such as RGD, YIGSR, IKVAV, andGFOGER peptides, are attached to PEG polymers to provide for celladhesion to the otherwise inert PEG hydrogel. In some embodiments, thehydrogels are biodegradable, e.g., by proteolytic degradation mechanismspresent in the natural ECM. For example, embodiments of the technologycomprise hydrogels crosslinked with (or otherwise incorporating)protease sensitive peptides. Some exemplary peptide sequences havingprotease sensitivity are derived from collagen (e.g.,matrix-metalloproteinase (MMP) sensitive sequence) and fibrin (e.g.,plasmin sensitive sequences). In some embodiments, protease-sensitivepeptides (e.g., comprising a matrix-metalloproteinase (MMP) sensitivesequence or a plasmin sensitive sequence) are incorporated into PEGhydrogels for degradation of the hydrogel, e.g., by the host enzymeactivities (see, e.g., Lutolf and Hubbell (2005) “Synthetic biomaterialsas instructive extracellular microenvironments for morphogenesis intissue engineering” Nat Biotechnol 23(1): 47-55). In sum, hydrogelsprovide a three-dimensional environment for the encapsulated cells ortissues (e.g., ovarian follicles), which promotes follicle function andsurvival in the device.

Some embodiments comprise use of a peptide that is a plasmin sensitivepeptide, e.g., for use as a degradable cross-linker in some embodimentsof the technology. Some embodiments comprise use of a peptide (e.g., aplasmin-sensitive peptide) that has the amino acid sequence:

(SEQ ID NO: 1) Ac-GCYK↓NSGCYK↓NSCG

In the amino acid sequence of the plasmin sensitive peptide, theN-terminal acetyl group is added to remove the electrical charge on thisterminal. The arrows indicate the protease cleavage sites. That is,embodiments comprise use of a peptide that has an amino acid sequenceaccording to:

(SEQ ID NO: 2) GCYKNSGCYKNSCGwith an N-terminal acetyl group and that is cleaved by a protease (e.g.,plasmin) between lysine and asparagine in the sequence, e.g., after thelysine at position 4 and/or after the lysine at position 10.

Some embodiments comprise use of a peptide that is sensitive to bothplasmin and MMP proteases, e.g., for use as a degradable cross-linker insome embodiments of the technology. Some embodiments comprise use of apeptide (e.g., a peptide that is both plasmin-sensitive andMMP-sensitive) that has the amino acid sequence:

(SEQ ID NO: 3) GCRDVPMS↓MRGGDRCGYK↓NSCG

In the amino acid sequence of the peptide that is both plasmin-sensitiveand MMP-sensitive, the arrows indicate the protease cleavage sites. Thatis, embodiments comprise use of a peptide that has an amino acidsequence according to:

(SEQ ID NO: 4) GCRDVPMSMRGGDRCGYKNSCGthat is cleaved by a protease (e.g., plasmin and/or MMP) between serineand methionine in the sequence at positions 8 and 9 and/or between thelysine and asparagine in the sequence at positions 18 and 19. Thispeptide sequence is sensitive to MMP and plasmin proteases and thuspresents a degradable cross-linker for encapsulated tissue and thatfinds use with several tissue types, e.g., tissues that comprise one orboth of a plasmin and/or MMP protease.

Accordingly, in some embodiments the technology provides animmunoisolation device comprising a degradable inner core comprisingcells; and a non-degradable outer shell encapsulating the degradableinner core. The technology is not limited in the types of cells providedin the inner core. For example, in some embodiments, the immunoisolationdevice comprises cells that are from an endocrine organ, e.g., from anovary. Further, the technology is not limited in the materials that areused to produce the inner core and the outer shell. In some exemplaryembodiments, the degradable inner core comprises a polyethylene glycoland, in some embodiments, the non-degradable outer shell comprises acrosslinked polyethylene glycol. Embodiments contemplate various typesof crosslinking of the inner core and outer shell; in some embodiments,the degradable inner core comprises a polyethylene glycol crosslinkedwith a degradable peptide, e.g., a degradable peptide comprising amatrix-metalloproteinase (MMP) sensitive sequence and/or a plasminsensitive sequence. In some embodiments, the inner core and/or the outershell comprises a polyethylene glycol hydrogel, e.g., a polyethyleneglycol vinyl sulfone hydrogel, e.g., a photo-polymerized polyethyleneglycol vinyl sulfone. In some embodiments, the immunoisolation devicecomprises polyethylene glycol at 2% to 15% (w/v) and/or the outer shellcomprises polyethylene glycol at 2% to 15% (w/v), e.g., 2%, 2.5%, 3%,3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or 15% PEG.

Some embodiments comprise use of a non-degradable PEG, e.g.,PEG-maleimide (PEG-Mal). Some embodiments comprise use of ahydrolytically-degradable PEG, e.g., PEG-acrylate (PEG-Ac).

As described herein, embodiments of the immunoisolation device comprisean inner core configured to allow the cells to grow and the outer shellis configured to allow exchange of metabolites with the environmentoutside the immunoisolation device and to protect the encapsulated cellsfrom immune recognition by the host immune system components outside theimmunoisolation device.

In certain embodiments of the immunoisolation device (e.g., embodimentsassociated with treating a female endocrine deficiency), theimmunoisolation device further comprises estrogen and/or progesteroneproduced by the cells. And, in some embodiments, the immunoisolationdevice further comprises a drug, e.g., an immunosuppressive drug.

In some embodiments, the immunoisolation device of claim furthercomprises polyvinylpyrrolidone.

Related embodiments provide a kit comprising a degradable PEG hydrogelprecursor solution and a non-degradable PEG hydrogel precursor solution,e.g., a degradable PEG vinyl sulfone hydrogel precursor solution and anon-degradable PEG vinyl sulfone hydrogel precursor solution. Some kitembodiments further comprise a buffer (e.g., HEPES buffer); aphotoinitiator; and/or a cross-linker.

Exemplary embodiments comprise a photo-polymerizable and/orphoto-polymerized PEG vinyl sulfone system. In some embodiments, thetechnology relates to a PEG precursor solution comprising 0.01 to 1%photoinitiator (e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4,0.5%, 0.6, 0.7, 0.8, 0.9, or 1.0% (w/v) photoinitiator). In someembodiments PEG precursor is irradiated (e.g., by ultraviolet light) for1 to 10 minutes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes ormore). In some embodiments, polymerization is initiated in the presenceof polyvinylpyrrolidone (PVP). For example, some embodiments of thetechnology provide a non-degradable PEG vinyl sulfone system comprising5% PEG, 0.4% photoinitiator, and 0.1% (v/v) PVP.

Further embodiments provide a method of treating a subject for anendocrine deficiency comprising implanting the immunoisolation device.For example, in some embodiments the cells of the immunoisolation deviceproduce a bioactive material (e.g., a hormone such as estrogen and/orprogesterone) for which the subject is deficient. Particular embodimentsrelate to treatment of a subject. In some embodiments, the subject is afemale who was treated for a reproduction cancer as a child. In someembodiments, the subject is a female who is in need of hormone therapyfor menopause.

In some embodiments, the technology relates to an immunoisolation devicecomprising a synthetic membrane, e.g., a bilayer comprising an innersemipermeable membrane made of polytetrofluoroethylene (PTFE) that islaminated to an outer membrane covered by a loose polyester mesh (e.g.,commercially available as THERACYTE, TheraCyte, Inc., Laguna Hills,Calif.) (e.g., a “TheraCyte pouch” or a “TheraCyte bag”). For example,some embodiments relate to devices comprising cells (e.g., endocrinecells, e.g., ovarian cells) placed in a synthetic membrane, e.g., forimmunoisolation of the cells, e.g., for transplantation into a host, andmethods relating to placing cells (e.g., endocrine cells, e.g., ovariancells) into a synthetic membrane pouch or bag and methods relating totransplanting the cells (e.g., endocrine cells, e.g., ovarian cells) inthe synthetic pouch or bag into a host.

In some embodiments, the technology provided herein provides for thesurvival and/or growth of transplanted cells in a host. For example, insome embodiments the technology provides for the survival and/or growthof transplanted cells for 1 to 30 days or more (e.g., for a number ofdays that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, or for morethan 30 days).

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is series of plots showing the swelling ratio of ND-PEG-VScomprising 0.5%, 0.4%, and 0.3% photoinitiator (FIG. 1A, FIG. 1B, andFIG. 1C, respectively) and 5% D-PEG-VS (FIG. 1D).

FIG. 2 is a plot showing the measured storage moduli of ND-PEG-VS (at 5%and 10%) and D-PEG-VS (5%).

FIG. 3 is a series of plots showing diffusion of varying dextran sizesover time in 5% (FIG. 3A) and 10% (FIG. 3B) ND-PEG-VS. FIG. 3C is a plotshowing the proportion of varying dextran sizes washed off the surfaceof the PEG hydrogels after a 24 hour soak relative to the dextranreleased over 24 hours.

FIG. 4 is a plot showing a decrease in FSH levels in ovariectomized miceafter implantation of ovaries encapsulated in TheraCyte® for a period of7 (lower line) and 30 (top line) days.

FIG. 5 is a bar plot showing the swelling ratios of PEG-acrylate(PEG-Ac), PEG-vinyl sulfone (PEG-VS), and PEG-maleimide (PEG-Mal)hydrogels.

FIG. 6 is a bar plot showing the swelling ratios (Qm) of D-PEG andND-PEG hydrogels.

FIG. 7 is a bar plot showing the storage modulus (G′) of D-PEG hydrogeland ND-PEG hydrogels formed with 3, 5, and 10 minute crosslinking times.

FIG. 8 is a plot showing the storage and loss moduli (G′ and G″,respectively) measured by rheology for PEG-acrylate (PEG-Ac), PEG-vinylsulfone (PEG-VS), and PEG-maleimide (PEG-Mal) hydrogels.

FIG. 9 is a bar plot showing the swelling ratios for PEG-acrylate(PEG-Ac), PEG-vinyl sulfone (PEG-VS), and PEG-maleimide (PEG-Mal)hydrogels incubated in 5 mM buffered and NaOH solutions at 37° C. for 30days.

FIG. 10 is a series of micrographs. (10A) ovarian tissue encapsulated inD-PEG; (10B) D-PEG in the subcutaneous space 30 days post implantation;(10C) retrieved D-PEG; (10D) ovarian tissue encapsulated in ND-PEG;(10E) ND-PEG at the time of sacrifice; (10F) retrieved D-PEG; (10G)ovarian tissue encapsulated in Dual-PEG; (10H) dual-PEG at the time ofsacrifice; (10I) retrieved dual-PEG after removal. Dotted circleindicates the localization of the hydrogel implanted in the mice. Whitearrows indicate encapsulated ovarian tissue. Gray arrows indicate theborder of D-PEG and black arrows indicate the border of ND-PEG.

FIG. 11 is a series of histological images of ovarian tissueencapsulated in D-PEG (11A) showing primordial follicles (*) in 7 dayimplants; (11B), (11C) showing secondary (***) and antralfollicles(****) in 30 and 60 day implants respectively; ND-PEG implantsshowing primary and secondary follicles (11D, 11E, 11F) after 7, 30, and60 days, respectively, and dual PEG implants showing secondary (11G) andantral (11H) follicles in 7 and 30 day implants respectively. (11I)Ovarian tissue encapsulated in dual PEG after 60 days of implantation.Magnification 10× (11D, 11E, 11H, 11I); 20× (11A, 11B, 11C, 11F, 11G).

FIG. 12 is a plot showing the survival and function of encapsulatedovarian tissue. Data were collected by monitoring the frequency of theestrous cycle in mice that received tissue implanted in ND-PEG, D-PEG,and dual PEG.

FIG. 13A is a bar plot showing FSH levels in mice before ovariectomy,after ovariectomy, and at week 4, and 60 days after implanting ovariantissue encapsulated in D-PEG.

FIG. 13B is a bar plot showing FSH levels in mice before ovariectomy,after ovariectomy, and at week 4, and 60 days after implanting ovariantissue encapsulated in ND-PEG.

FIGS. 14A and 14B are macroscopic images showing allogeneic ovariantissue encapsulated in D-PEG (left images) and retrieved at the time ofsacrifice (right images).

FIGS. 15A and 15B are images showing that implantation of allogeneicovarian tissue without encapsulation results in rejection andelimination of the implanted tissue after 28 days of implantation;analysis of the images indicated that no ovarian follicles at any stagewere found in the implanted tissue. The images were acquired at amagnification of 5× (15A) and 20× (15B).

FIG. 16A is a plot of percentage of estrous cycling mice afterimplantation of allogeneic ovary tissue without encapsulation in animmunoisolation device.

FIG. 16B is a plot of percentage of estrous cycling mice afterimplantation of allogeneic ovary tissue a D-PEG immunoisolation device.

FIG. 16C is a plot of percentage of estrous cycling mice afterimplantation of allogeneic ovary tissue in a Theracyte immunoisolationdevice.

FIG. 16D is a plot of percentage of estrous cycling mice afterimplantation of allogeneic ovary tissue in a Dual PEG immunoisolationdevice.

FIG. 17 shows flow cytometry data collected to evaluate the immunereaction towards the implanted allogeneic tissue without the device(allogeneic control), healthy mice without the device (sham surgery),and encapsulated tissue (allogeneic ovarian tissue encapsulated in dualPEG). Pre-TX indicates pre-implantation. Without immunoisolation, therecipients developed IgG 21 days after receiving the allograft (enclosedby the rectangle). Mice that received allograft encapsulated in dual PEGhydrogel presented no antibodies up to 60 days post implantation,similar to the negative controls. Plots were obtained and meanfluorescence intensities (MFI) in the APC-channel determined with FlowJosoftware. Rectangles indicate thymocytes bound to IgG.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

Provided herein is a technology for restoring a biological function(e.g., an endocrine function) in a subject. For example, in someembodiments the technology relates to restoring ovarian function, e.g.,in young women with POF or for treating women during menopause. Inparticular embodiments, the technology relates to transplantation ofovarian tissue with minimized or eliminated host rejection. As describedherein, embodiments of the technology provide a dual hydrogel constructthat is used to encapsulate transplanted ovarian follicles that secretesex hormones in the host, e.g., in response to circulatinggonadotropins. Accordingly, embodiments of the technology find use inimproving (e.g., establishing a normal) physiological endocrine ovarianfunction in the transplant host.

An allogeneic system to provide endocrine support for ovarian tissuetransplantation has yet to be established. While transplantation ofovarian follicles has similarities to transplantation of islet cells totreat diabetes (see, e.g., (Garcia (2014) Ann Biomed Eng 42(2): 312-22;Shea et al. (2011) Diabetologia 54 (10): 2494-505; Anseth et al. (2008)Cell Transplant 16(10): 1049-57)), the transplantation of ovarianfollicles is associated with its own set of unique challenges that havenot been addressed by previous technologies. Follicles have a similarinitial size to islets and secrete sex hormones (estradiol andprogesterone) in response to a circulating stimulus. However, unlikeislets, follicles expand and contract as they undergo structural andfunctional changes during the menstrual cycle, which is a feature notsupported by static microencapsulation materials. Furthermore, folliclesare avascular and relatively resistant to hypoxia, allowing them tosurvive when implanted as larger structures, which provides an advantagecompared to highly vascularized islets. Accordingly, the technologyprovides a hydrogel that has an immunoisolating exterior (“outer shell”)and a degradable core (“inner core”) capable of supporting prolongedsurvival and restoration of endocrine function following allogeneicovarian transplantation.

In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control. The section headings used herein arefor organizational purposes only and are not to be construed as limitingthe described subject matter in any way.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein, the term “polymer” means any compound that is made up oftwo or more monomeric units covalently bonded to each other, where themonomeric units may be the same or different, such that the polymer maybe a homopolymer or a heteropolymer. Representative polymers includepolyamides (e.g., such as polypeptides), poly-N-substituted glycines(polypeptoids), polysaccharides, polyethylene glycol (PEG), plastics,polynucleotides (e.g., nucleic acids), and the like, where the polymersmay be naturally occurring, non-naturally occurring, or synthetic.

As used herein, the term “poly(ethylene glycol)”, abbreviated “PEG”,refers to a synthetic polymer of ethylene glycol. PEG is water-solubleand can be modified with various functional groups that allow one totailor its chemistry, physical, and biological properties.

As used herein, the term “polypeptides” includes proteins and fragmentsthereof (e.g., peptides). In some embodiments, polypeptides aredisclosed as amino acid residue (or monomer) sequences. Those sequencesare written left to right in the direction from the amino to the carboxyterminus. In accordance with standard nomenclature, amino acid residuesequences are denominated using either a three letter code or a singleletter code as indicated as follows: Alanine (Ala, A), Arginine (Arg,R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine(His, H), Isoleucine I), Leucine (Leu, L), Lysine (Lys, K), Methionine(Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S),Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine(Val, V). In addition, a polypeptide can include non-standard and/ornon-naturally occurring amino acids or post-translationally modifiedamino acids such as hydroxylated amino acids, as well as other aminoacids that may be found in phosphorylated proteins in organisms such as,but not limited to, animals, plants, insects, protists, fungi, bacteria,algae, single-cell organisms, and the like. The non-standard amino acidsinclude, but are not limited to, selenocysteine, selenomethionine,pyrrolysine, gamma-aminobutyric acid, carnitine, ornithine, citrulline,homocysteine, hydroxyproline, hydroxylysine, sarcosine, and the like.The non-naturally occurring amino acids include, but are not limited to,trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,trans-4-hydroxyproline, N-methylglycine or other N-substituted glycines,beta-amino acids, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitro-glutamine, homoglutamine, pipecolicacid, thiazolidine carboxylic acid, dehydroproline, 3- and4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and4-fluorophenylalanine.

As used herein, the term “polynucleotide” generally refers to anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, the term“polynucleotide” as used herein refers to, among others, single-strandedand double-stranded DNA, DNA that is a mixture of single-stranded anddouble-stranded regions, single-stranded and double-stranded RNA, andRNA that is a mixture of single-stranded and double-stranded regions,hybrid molecules comprising DNA and RNA that may be single-stranded or,more typically, double-stranded or a mixture of single-stranded anddouble-stranded regions. The term “polynucleotide” encompasses the terms“nucleic acid”, “nucleic acid sequence”, and “oligonucleotide”. Inaddition, the term “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term “polynucleotide” includes nucleic acids thatcomprise one or more modified bases. Thus, nucleic acids with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, nucleic acids comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, arepolynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically, or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells.

As used herein, the terms “treatment”, “treating”, and “treat” refer toacting upon a disease (e.g., cancer), disorder, or condition (e.g.,biological deficiency or discomfort, e.g., one or more symptoms ofmenopause) with an agent to reduce or ameliorate the pharmacologicand/or physiologic effects of the disease, disorder, or condition and/orits symptoms. “Treatment,” as used herein, covers any treatment of adisease, disorder, or condition in a host (e.g., a mammal, typically ahuman or non-human animal of veterinary interest), and includes: (a)reducing the risk of occurrence of a disease, disorder, or condition ina subject determined to be predisposed to the disease, disorder, orcondition, but not yet diagnosed as having the disease, disorder, orcondition; (b) impeding the development of a disease, disorder, orcondition; (c) relieving a disease, disorder, or condition, e.g.,causing regression of the disease, disorder, or condition and/orrelieving one or more symptoms of the disease, disorder, or condition;(d) ameliorating, reducing, or reversing one or more symptoms of adisease, disorder, or condition and/or ameliorating, reducing, orreversing one or more symptoms resulting from a treatment for thedisease, disorder, or condition; and (e) providing or restoring normal(or near normal, adequate, sufficient, essentially normal, and/oreffectively normal) or improved biological processes in a subject, e.g.,a subject who has impaired or damaged biological processes due to adisease, disorder, or condition and/or from treatment of a disease,disorder, or condition. “Treatment” is also meant to encompass providinga pharmacologic effect in a normal subject or in a subject in theabsence of a disease, disorder, or condition. For example, “treatment”encompasses providing for improved, enhanced, or desirable effects inthe subject (e.g., reduction of tumor load, reduction of symptoms,improved or normal growth and development, extension of a period of apatient's apparent, functional health, etc.).

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refers to completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease.

The term “attached” or the phrases “interacts with” and “associatedwith” refers to a stable physical, biological, biochemical, and/orchemical association. In general, association can be chemical bonding(e.g., covalently or ionically), a biological interaction, a biochemicalinteraction, and in some instances a physical interaction. Theassociation can be a covalent bond, a non-covalent bond, an ionic bond,a metal ion chelation interaction, as well as moieties being linkedthrough interactions such as, but not limited to, hydrophobicinteractions, hydrophilic interactions such as hydrogel bonding,charge-charge interactions, π-stacking interactions, combinationsthereof, and like interactions.

The term “cancer”, as used herein, shall be given its ordinary meaning,as a general term for diseases in which abnormal cells divide withoutcontrol and form cancer or neoplastic cells, tissues, or tumors. Theterm cancer can include cancer cells and/or precancerous cells. Inparticular, and in the context of the embodiments of the presentdisclosure, cancer refers to ovarian cancer and cancers of the femalereproductive organs and system. Cancer cells can invade nearby tissuesand can spread through the bloodstream and lymphatic system to otherparts of the body. There are several main types of cancer, for example,carcinoma is cancer that begins in the skin or in tissues that line orcover internal organs. Sarcoma is cancer that begins in bone, cartilage,fat, muscle, blood vessels, or other connective or supportive tissue.Leukemia is cancer that starts in blood-forming tissue such as the bonemarrow, and causes large numbers of abnormal blood cells to be producedand enter the bloodstream. Lymphoma is cancer that begins in the cellsof the immune system.

When normal cells lose their ability to behave as a specified,controlled, and coordinated unit, a tumor may be formed. Generally, asolid tumor is an abnormal mass of tissue that usually does not containcysts or liquid areas (although some brain tumors do have cysts andcentral necrotic areas filled with liquid). A single tumor may even havedifferent populations of cells within it, with differing processes thathave gone awry. Solid tumors may be benign (not cancerous) or malignant(cancerous). Different types of solid tumors are named for the type ofcells that form them. Examples of solid tumors are sarcomas, carcinomas,and lymphomas. Leukemias (cancers of the blood) generally do not formsolid tumors.

Representative cancers include, but are not limited to, bladder cancer,breast cancer, colorectal cancer, endometrial cancer, head and neckcancer, leukemia, lung cancer, lymphoma, melanoma, non-small-cell lungcancer, ovarian cancer, prostate cancer, testicular cancer, uterinecancer, cervical cancer, thyroid cancer, gastric cancer, brain stemglioma, cerebellar astrocytoma, cerebral astrocytoma, glioblastoma,ependymoma, Ewing's sarcoma family of tumors, germ cell tumor,extracranial cancer, Hodgkin's disease, leukemia, acute lymphoblasticleukemia, acute myeloid leukemia, liver cancer, medulloblastoma,neuroblastoma, brain tumors generally, non-Hodgkin's lymphoma,osteosarcoma, malignant fibrous histiocytoma of bone, retinoblastoma,rhabdomyosarcoma, soft tissue sarcomas generally, supratentorialprimitive neuroectodermal and pineal tumors, visual pathway andhypothalamic glioma, Wilms's tumor, acute lymphocytic leukemia, adultacute myeloid leukemia, adult non-Hodgkin's lymphoma, chroniclymphocytic leukemia, chronic myeloid leukemia, esophageal cancer, hairycell leukemia, kidney cancer, multiple myeloma, oral cancer, pancreaticcancer, primary central nervous system lymphoma, skin cancer, small-celllung cancer, among others.

A tumor can be classified as malignant or benign. In both cases, thereis an abnormal aggregation and proliferation of cells. In the case of amalignant tumor, these cells behave more aggressively, acquiringproperties of increased invasiveness. Ultimately, the tumor cells mayeven gain the ability to break away from the microscopic environment inwhich they originated, spread to another area of the body (with a verydifferent environment, not normally conducive to their growth), andcontinue their rapid growth and division in this new location. This iscalled metastasis. Once malignant cells have metastasized, achieving acure is more difficult. Benign tumors have less of a tendency to invadeand are less likely to metastasize.

As used herein “administration” refers to introducing a compound into asubject.

As used herein, the term “subject”, “host”, or “organism” includeshumans and mammals (e.g., cats, dogs, horses, etc.). In someembodiments, subjects are mammals, particularly primates, especiallyhumans. For veterinary applications, a wide variety of subjects issuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.

As used herein, the term “hydrogel” refers to a substance formed when anorganic polymer (natural or synthetic) is cross-linked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure that entraps water molecules to form a gel. As used herein,the term “biocompatible hydrogel” refers to a polymer that forms a gelthat is not toxic to living cells and allows sufficient diffusion ofoxygen and nutrients to entrapped cells to maintain viability.

As used herein, the term “biocompatible” generally refers to a materialand any metabolites or degradation products thereof that are generallynon-toxic to the recipient and do not cause any significant adverseeffects to the subject.

As used herein, phosphate buffered saline, abbreviated “PBS”, andDulbecco's phosphate buffered saline, abbreviated “DPBS”, are bufferedsalines used in biological studies. PBS and DPBS are used in researchinvolving cells. The ion concentration and osmolarity of PBS and DPBSare isotonic, that is, compatible with the human body. In someembodiments, these buffers provide and preserve a stable pH of 7.2-7.6.There is no significant difference between PBS and DPBS. Both of themcontain sodium phosphate, sodium chloride, and, when required, potassiumphosphate and potassium chloride. In some embodiments, preparations ofPBS or DPBS may or may not contain calcium and magnesium. PBS and DPBShave numerous applications because they are not noxious to cells. BothPBS and DPBS can be used to rinse instruments or containers contaminatedwith cells. Also, both of them can be used in diluting substances.

As used herein, the terms “degradable” and “biodegradable” generallyrefer to a material that degrades or erodes by hydrolysis or enzymaticaction under physiologic conditions to smaller units or chemical speciesthat are capable of being metabolized, eliminated, or excreted by thesubject.

As used herein, the “degradation rate” refers to a rate relating thenumber of smaller units or chemical species that are produced bybiodegradation or degradation of a material as a function of time.

As used herein, the “degradation time” refers to the time required toproduce a threshold number of smaller units or chemical species bybiodegradation or degradation of a material and is a function of polymercomposition and morphology.

As used herein, the term “non-degradable” refers to a material that isnot “degradable” or that is substantially or effectively less degradablethan a degradable material (e.g., has a substantially or effectivelylower degradation rate or a substantially or effectively longerdegradation time).

As used herein, the term “mammalian cell” refers to any cell derivedfrom a mammalian subject suitable for transplantation into the same or adifferent subject. The cell may be syngeneic, xenogeneic, autologous, orallogeneic. The cell can be a primary cell obtained directly from amammalian subject. The cell may also be a cell derived from the cultureand expansion of a cell obtained from a subject. For example, the cellmay be a stem cell. Immortalized cells are also included within thisdefinition. In some embodiments, the cell has been geneticallyengineered to express a recombinant protein and/or nucleic acid.

As used herein, the term “transplant” refers to the transfer of a cell,tissue, or organ to a subject from another source. The term is notlimited to a particular mode of transfer. Encapsulated cells may betransplanted by any suitable method, such as by injection or surgicalimplantation.

As used herein, the term “autologous” refers to a transplantedbiological substance taken from the same individual.

As used herein, the term “xenogeneic” refers to a transplantedbiological substance taken from a different species. As used herein, theterm “xenogeneic transplantation” refers to the transplantation ofliving cells, tissues, or organs from one species to another. Suchcells, tissues, or organs are called “xenografts” or “xenotransplants”.Both allotransplantation (e.g., a same-species transplant) andxenotransplantation can cause rejection of the graft because the immunesystem of the host recognizes the transplant as foreign (e.g., as“non-self”). In addition to rejection, disease transmission(“xenozoonosis”) and permanent alteration to the host genetic code arecauses for concern. Accordingly, the use of immunoisolating devicesprovides a technology to prevent, eliminate, and/or minimize rejectionof a graft and/or to prevent, eliminate, and/or minimize the risk ofdisease transmission.

As used herein, the term “allogeneic” refers to a transplantedbiological substance taken from a different individual of the samespecies. Accordingly, as used herein, the term “allogeneictransplantation” or “allotransplantation” refers to the transplantation(e.g., of a cell, tissue, or organ) to a recipient from a geneticallynon-identical donor of the same species. The transplant is called anallograft, allogeneic transplant, or homograft. Most human tissue andorgan transplants are allografts because humans genetically differ fromeach other. Similarly, transplantation of a tissue between differentstrains of mice is termed an allogeneic transplantation. An immuneresponse against an allograft, termed rejection, will arise in healthyindividuals without immune suppression.

As used herein, the term “isogeneic transplantation” or “syngraft” is agraft between genetically identical individuals, typically betweenidentical twins or between animals of a single highly inbred strain.This type of graft typically does not provoke the immune system and doesnot cause rejection.

As used herein, the term “endocrine system” refers to the collection ofcells and tissues of an organism that secrete hormones directly into theblood to control physiological and behavioral activities of theorganism. The endocrine system comprises a series of glands that producemolecules called hormones. A number of glands that signal to each otherin a sequence are usually referred to as an axis, for example, thehypothalamic-pituitary-gonadal (HPG) axis that in a female connects theglands involved in regulating the ovarian function. Reproductiveendocrine function is mediated by sex hormones, such as estradiol andprogesterone. Besides the effect of the sex hormones on the reproductiveorgan, they have other functions, such as metabolism, fat storage, bloodvessel and skin maintenance, protein synthesis, prevention of boneresorption, and muscle degeneration.

As used herein, the term “endogenous” as it relates to an organism orbiological system refers to a substance, molecule, etc. produced orsynthesized within the organism or biological system.

As used herein, the term “endogenous hormones” as it relates to anorganism or biological system refers to hormones produced or synthesizedwithin the organism or biological system. For example, in femalesestradiol is produced in special structures called ovarian follicles.Follicles produce estradiol in response to other hormones that regulateovarian function.

As used herein, the terms “epiphyses” and “epiphyseal growth plate”refer to features of a bone. Bone is a living tissue comprising aprotein (collagen) matrix upon which calcium salts are deposited. Agrowing bone is described by the ends, or epiphyses, and the shaft. Theportion of each epiphysis in contact with the shaft is a plate ofactively proliferating cartilage (connective tissue composed of collagenand other fibrous proteins) called the epiphyseal growth plate. Lineargrowth of the shaft can continue as long as the epiphyseal growth platesexist, but cease when the growth plates are converted to bone as aresult of hormonal influences at puberty. This is known as epiphysealclosure and occurs at different times in different bones.

As used herein, the term “estrogen” refers to a class of steroidhormones secreted by the ovaries. For example, estradiol is apredominant estrogen in the plasma. Estradiol is produced and secretedfrom ovaries and it plays a key role in puberty, providing a hormonalmilieu for physical and psychosocial development. Estradiol isresponsible for the development of the female appearance, bone growth,and brain development. Simultaneously, increases in estradiol levelsduring puberty stimulate other growth hormones that lead to the pubertalgrowth spurt. The hypothalamus, pituitary gland, and the ovary interactalong the “HPG axis”. The pulsatile release of Gonadotropin ReleasingHormone (GnRH) from the hypothalamus stimulates the secretion ofLuteinizing hormone (LH) and Follicle stimulating hormone (FSH) from thepituitary gland in the brain. FSH directly stimulates granulosa cells inthe growing follicles to secrete estradiol. LH stimulates theca cells inthe follicle to produce precursors of estradiol to increase itsproduction. HPG axis is a loop that is tightly regulated by the secretedhormones.

As used herein, the term “exogenous” as it relates to an organism orbiological system refers to a substance, molecule, etc. originating fromoutside an organism or biological system.

As used herein, the term “exogenous hormone” as it relates to anorganism or biological system refers to a hormone originating fromoutside the organism or biological system. Exogenous estrogen is asynthetic analog of the estradiol and can mimic the function ofendogenous estradiol.

As used herein, the term “follicle” or “ovarian follicle” refers to thefunctional unit of the ovary. It contains a germ cell that is future todevelop into an egg, surrounded by multiple layers of supportive cells,called granulosa cells. In the ovaries, granulosa cells synthesize andsecrete estradiol in response to the hormones that control the ovarianfunction. Theca cells build the outside layer of the follicle. Thecacells produce androgens, which are the precursors for estradiol producedby granulosa cells.

As used herein, the term “follicular stimulating hormone”, abbreviated“FSH”, refers to a hormone that is secreted from the pituitary gland asa result of hypothalamus stimulation. FSH acts on ovaries and stimulatesestradiol secretion and follicle growth.

As used herein, the term “folliculogenesis” refers to a process thatdescribes the maturation of the ovarian follicle, a densely packed shellof somatic cells that surround a germ cell. Folliculogenesis describesthe progression of small immature follicles to a mature follicle readyfor ovulation. Hormones secreted from hypothalamus (GnRH) and pituitarygland (FSH and LH) regulate the process of follicle development. Inresponse to hormonal stimulation follicles produce estradiol andprogesterone that regulate the hormone production in the brain in aseries of positive and negative feedback mechanisms. The levels of allhormones in the HPG axis cyclically fluctuate.

As used herein, the term “gamete” refers to a cell that fuses withanother cell during fertilization in organisms that reproduce sexually.In a female, the gamete is as “egg”.

As used herein, the term “germ cell” refers to a cell that gives rise toa female gamete (egg) or a male gamete (sperm).

As used herein, the term “hormone” refers to a substance released fromendocrine tissue into the bloodstream where it travels to a targettissue to generate a response. Hormones regulate various humanfunctions, including metabolism, growth and development, tissuefunction, sleep, and mood. The term hormone as used herein alsoencompasses natural or synthetic molecules having the same or similarbioactive properties as a hormone released by endocrine tissue; andencompasses derivatives of natural and synthetic hormones and naturaland synthetic molecules having the same or similar bioactive propertiesas a hormone released by endocrine tissue.

As used herein, the term “gonadotropin releasing hormone”, abbreviated“GnRH”, is the first hormone in the axis between hypothalamus, pituitarygland, and ovary. GnRH stimulates the secretion of the hormones from thepituitary gland (FSH and LH), which in turn control the ovarianfunction.

As used herein, the term “immunoisolation” or “immune isolation” refersto a strategy used to protect a therapeutic, such as implanted cells ortissue, from being rejected by a donor. By providing a barrier aroundthe implanted cells or tissue, an immunoisolation device allows thepassage of nutrients and oxygen into the device to support the survivalof implanted cells or tissue and prevents the passage of immune cellsand antibodies into the device, thus eliminating or minimizing rejectionof the implanted cells or tissue by the host immune system.Immunoisolation of implanted cells or tissue allows foreign grafts tosurvive for extended, often indefinite intervals.

As used herein, the term “in vitro” refers to an environment outside aliving organism. In science this term refers to experiments performed inan artificial or synthetic environment.

As used herein, the term “in vivo” refers to within a living organism.In science this term refers to experiments performed in an animal modelor in humans.

As used herein, the term “luteinizing hormone”, abbreviated “LH” refersto a hormone that is secreted from the pituitary gland as a result ofhypothalamus stimulation. LH acts on theca cells in the follicles tostimulate the production of the precursors of estradiol.

As used herein, the term “primordial follicles” refers to immature andundeveloped stage of the follicles. These follicles contain one germcell, which is surrounded by several somatic cells. The primordialfollicles constitute the majority of the ovarian reserve at any age.

As used herein, the term “thrombosis” refers to the formation of a bloodclot (“thrombus”) inside a blood vessel, obstructing the flow of bloodthrough the circulatory system. When a thrombus is significantly largeenough to reduce the blood flow to a tissue, oxygen deprivation canoccur and metabolic products can accumulate. A larger thrombus causing amuch greater obstruction to the blood flow may result in anoxia, thecomplete deprivation of oxygen and tissue death.

As used herein, the term “biological communication” refers to theability of a biological component to communicate with another biologicalcomponent, e.g., by exchange of communicating substances such asmetabolites, catabolites, proteins, nucleic acids, small molecules(e.g., hormones), lipids, etc. with the biological component. A firstbiological component in biological communication with a secondbiological component is exposed to communicating substances producedand/or secreted by the second biological component. As used herein, a“biological component” is not limited by size or scale and thus may be amolecule, biological structure, organelle, cell, tissue, organ, system,or organism.

DESCRIPTION

Transplant rejection is an adaptive immune response that occurs viacellular immunity (mediated by killer T cells) as well as humoralimmunity (mediated by activated B cells secreting antibody molecules),along with an innate immune response mediated by phagocytic cells andsoluble immune proteins. Cellular immunity protects the body byactivating antigen-specific cytotoxic T-lymphocytes that are able toinduce apoptosis in body cells displaying epitopes of foreign antigen ontheir surface, activating macrophages and natural killer cells, andstimulating cells to secrete a variety of cytokines that influence thefunction of other cells involved in adaptive immune responses and innateimmune responses. Accordingly, provided herein is technology forminimizing and/or eliminating rejection of transplanted cells, e.g.,ovarian cells.

In some embodiments, the technology comprises use of donor ovariantissue encapsulated in an immunoisolating device. This technology findsuse in improving and/or restoring ovarian endocrine function andcontrolling host immunity, e.g., to restore ovarian endocrine function,for example, in young women with POF. The technology provides animmunoisolating device that is adapted for the particularcharacteristics of certain tissue types, e.g., ovarian tissue, andcomprises well-characterized biomaterials. Accordingly, the technologyprovides a new, clinically relevant therapy to improve or restoreovarian endocrine function (e.g., in young women and girls with POF) byimproving or restoring a physiological balance of the HPG axis thatexists in healthy women, which is difficult to achieve with exogenouspharmacological treatments.

Immunoisolation

Immunoisolation is a technique to protect transplanted tissue fromrejection by “hiding” the transplant from the recipient immune system.Immunoisolation approaches include use of semipermeable membranes,microencapsulation, and macroencapsulation technologies to provide forthe diffusion of nutrients and small molecules, while preventing freeexchange of cells. In some embodiments, semisolid hydrogels find use. Insome embodiments, an encapsulation device comprises an inert and durablepouch comprising a semipermeable membrane system.

The field of ovarian tissue transplantation for restoring fertility andovarian endocrine function in sterile women has progressed. However, aremaining concern relating to safe and ethical autotransplantation incancer survivors is avoiding the risk of re-introduction of malignantcells, which could lead to recurrence of the primary disease afterreimplantation. Multiple research groups have demonstrated that ovariantissue from leukemia patients is positive for malignant cells in >50% ofcases. Hematological malignancies, which contribute to 30% of allchildhood cancers, are of particular concern, because leukemia cells canbe found in any organ and have the potential to reseed the cancer whenthe ovarian tissue is transplanted back to the patient. Thus,transplanting the ovarian construct in an immunoisolating device notonly protects the follicles from host immune rejection, but alsoprotects the host from potential pathogens and cancer cells entering thebody. This approach also extends the pool of potential ovarian tissuedonors to xenografts and allografts.

In some embodiments, the technology provides an encapsulation device toprovide immune isolation for transplanted cells placed within theencapsulation device, e.g., the technology provides an immunoisolationdevice. The immunoisolation device allows the cells to exchangehormones, metabolites, catabolites, and other biologically activesubstances (except immune components) with the body of the host, butprotects the transplanted cells from immune recognition and immunerejection by the host.

Encapsulation/Immunoisolation Devices

In some embodiments, the technology relates toencapsulation/immunoisolation devices. For instance, in some embodimentsthe encapsulating device is a two-layer encapsulating device thatcomprises an “outer shell” and an “inner core”. In particularembodiments, the outer shell of the encapsulating device is preparedwith a non-degradable (ND) substance (e.g., a non-degradable hydrogel,e.g., comprising a first PEG (e.g., a PEG crosslinked by exposure toultraviolet radiation (e.g., crosslinked with a degradable (e.g.,proteolytically degradable) crosslinker))) and the inner core isprepared with a degradable substance (e.g., a degradable hydrogel, e.g.,comprising a second PEG (e.g., a PEG crosslinked by exposure toultraviolet radiation)). The non-degradable outer shell of this designprovides immune isolation of a therapeutic (e.g., transplanted cells,e.g., transplanted ovary cells) placed within the inner core and, inembodiments in which the therapeutic comprises cells or tissues, thedegradable inner core allows the implanted cell or tissue to expand asit grows, e.g., by the secretion of enzymes that degrade the inner core(e.g., proteases).

In some embodiments, the two-layer encapsulating device comprises twopoly(ethylene glycol) (PEG) hydrogels, e.g., a first PEG hydrogel thatprovides a degradable inner core and a second PEG hydrogel that providesa non-degradable outer shell. Therapeutics (e.g., tissue, e.g., ovariantissue) are implanted in the inner core of embodiments of the two-layerPEG encapsulating devices to provide immunoisolation for thetherapeutics implanted within the devices.

In some embodiments, the inner core comprises fibrin (e.g., a degradablefibrin). In some embodiments, the inner core comprises fibrin (e.g., adegradable fibrin) and PEG (e.g., a degradable PEG).

In some embodiments, the inner core further comprises a hormone, e.g.,in some embodiments the inner core comprises cells and a hormone. Insome embodiments, the inner core comprises ovarian cells and a sexhormone, e.g., a progesterone and/or an estrogen and/or anti-mullerianhormone (AMH). In some embodiments, the inner core comprises estrogenalone, estrogen plus progesterone, or estrogen plus progestin, which isa synthetic hormone with effects similar to those of progesterone. Thetechnology is not limited in the source of the sex hormone, e.g., thesource may be a plant, animal, or recombinant organism. The sex hormonemay be a bio-identical hormone, a synthetic hormone, or a hormoneisolated from a natural source. In some embodiments, the sex hormone isan estropipate, an estradiol, an estrogen, a conjugated estrogen; amedroxyprogesterone; a norethindrone; a drospirenone; a levonorgestrel;a norgestimate; and/or a bazedoxifene.

In some embodiments, the compositions are fabricated into syntheticorgans, such as a synthetic ovary containing encapsulated ovarian cells.In some of these embodiments, the cells are encapsulated in a singlehydrogel compartment. In other embodiments, the composition contains aplurality of microencapsulated cells dispersed or encapsulated in abiocompatible structure.

In some embodiments, the technology comprises use of PEG and crosslinkedPEG (e.g., to produce degradable PEG vinyl sulfone hydrogels,non-degradable PEG vinyl sulfone hydrogels, and dual PEG hydrogelscomprising degradable PEG vinyl sulfone hydrogels and non-degradable PEGvinyl sulfone hydrogels, as described below). Crosslinking PEG usingultraviolet light has been used to prepare PEG-based hydrogels. However,some PEG precursors are hydrolytically degradable, such as PEG-acrylate.Thus, while PEG-acrylate readily forms hydrogels upon addition of photoinitiators and irradiation, the presence of an ester bond in the polymerbackbone makes these hydrogels hydrolytically unstable and degradablewhen immersed in an aqueous environment, such as a live organism. Someembodiments comprise use of a non-degradable PEG that is PEG-maleimide.

Accordingly, the technology relates to a hydrogel system that is notsensitive to hydrolytic degradation and that is degraded by proteasessecreted by live cells (e.g., that is susceptible to a cell-driven(proteolytic) degradation). For example, in some embodiments 4-armPEG-VS finds use in the technology. However, prior to the development ofthe technology provided herein, a photo-polymerization protocol for4-arm PEG-VS had not been developed. Accordingly, the development of thetechnology provided herein was associated with developing aphoto-polymerized PEG-VS system, e.g., by testing varying Irgacure 2959concentrations (e.g., including but not limited to 0.05, 0.3, 0.4, 0.5%(w/v)), a range of irradiation times, and polymerization in the presenceof polyvinylpyrrolidone (PVP). After testing many combinations andperforming preliminary characterization experiments, some embodiments ofthe technology provide a ND-PEG-VS system comprising 5% PEG, 0.4%photoinitiator, and 0.1% (v/v) PVP. Furthermore, development of thetechnology was associated with developing associated testing methods tocharacterize the hydrogels and to collect data describing the hydrogels.For example, methods were developed to quantify dextran release fromgels by dissolving a dextran in a PEG precursor solution (e.g., at aconcentration of 1 mg/ml), gelling the PEG to produce a hydrogel, andthen measuring the release of dextran from the hydrogel. During thedevelopment of embodiments of the technology, it was discovered thatinclusion of dextran hindered gel formation and gels did not form usingthe aforementioned ND-PEG-VS protocol. Thus, experiments were conductedto develop alternative technologies, e.g., by soaking gels in a dextransolution, e.g., for 24 hours, and then left in D-PBS to quantify dextranrelease.

In some embodiments, an 8-arm PEG-VS (e.g., a 40 kDa 8-arm PEG-VS) isused for the outer shell (e.g., in some embodiments the outer shellcomprises 8-arm PEG-VS (e.g., 40 kDa 8-arm PEG-VS), e.g., cross-linked8-arm PEG-VS (e.g., cross-linked 40 kDa 8-arm PEG-VS)). In someembodiments, a 4-arm PEG-VS (e.g., a 20 kDa 4-arm PEG-VS) is used forthe outer shell (e.g., in some embodiments the outer shell comprises4-arm PEG-VS (e.g., 20 kDa 4-arm PEG-VS), e.g., cross-linked 4-armPEG-VS (e.g., 20 kDa 4-arm PEG-VS)). Without being bound by theory, itis contemplated that a lower molecular weight PEG produces a tighternetwork, further restricting the passage of host immune cells throughthe outer shell without affecting the exchange of nutrients andhormones.

Polyethylene Glycol (PEG)

PEG hydrogel is a synthetic multifunctional hydrophilic polymer.PEG-based hydrogels are not immunogenic and have biocompatible chemistrywithin physiological conditions. PEG provides a synthetic matrix thatprovides for controlling the degradation, stiffness, and pore sizecharacteristics of the encapsulation device. Accordingly, embodiments ofthe technology comprise the use of polyethylene glycol (PEG). PEG, alsoknown as polyethylene oxide (PEO) or polyoxyethylene (POE), is a polymerof ethylene oxide (e.g., a polyether) and has a molecular formulaaccording to (1) or (2) below:

H—(O—CH₂—CH₂)_(n)—OH  (1)

C_(2n)H_(4n+2)O_(n+1)  (2)

PEG is a versatile polymer prepared by polymerization of ethylene oxide.Various production methods produce PEG having a wide range of molecularweights (e.g., from 300 g/mol to 10,000,000 g/mol; e.g., from 100daltons to 100 kilodaltons), chain lengths, and geometries (e.g.,linear, branched, star, multi-arm, comb, etc.). In particular, branchedPEG has 3 to 10 PEG chains linked to a central core group, star PEG has10 to 100 PEG chains linked to a central core group, and comb PEG hasmultiple PEG chains linked to a linear polymer backbone.

The different sizes and geometrical forms of PEG are produced using aninitiator to initiate the polymerization of the ethylene oxide monomers.The most common initiator is a monofunctional methyl ether PEG, ormethoxypoly(ethylene glycol), abbreviated mPEG. Further, PEG isavailable in a wide range of purities, e.g., in some embodiments the PEGis a polydisperse PEG having a broad range of molecular weights orlengths or is a polydisperse PEG having a narrow range of molecularweights or lengths. In some embodiments, the PEG is monodisperse (e.g.,uniform PEG or discrete PEG), e.g., the PEG has a very high purity,e.g., the PEG has a molecular weight or chain length that ismonodisperse, uniform, or discrete. Very high purity PEG is crystalline.

PEG is often described using a number that indicates the averagemolecular weight of the PEG polymers. For example, a PEG with n=9 wouldhave an average molecular weight of approximately 400 daltons and wouldbe described by the name “PEG 400”. Most PEG preparations comprisemolecules with a distribution of molecular weights (e.g., a polydispersePEG). The size distribution is typically characterized by the weightaverage molecular weight (M_(w)) and its number average molecular weight(M_(n)), the ratio of which is called the polydispersity index(M_(w)/M_(n)). M_(w) and M_(n) can be measured by mass spectrometry.

PEGylation is the act of covalently coupling a PEG structure to anotherlarger molecule, for example, a therapeutic protein, which is thenreferred to as a PEGylated protein.

PEG is widely soluble, e.g., in water, methanol, ethanol, acetonitrile,benzene, and dichloromethane, and PEG is insoluble in diethyl ether andhexane.

Accordingly, PEG provides a versatile and “tunable” material for theproduction of devices described herein. For example, one can control thestiffness, size exclusion properties, and degradability of PEG hydrogelsby controlling the PEG concentration, cross-linking, and molecularweight and geometry of the PEG used to form the hydrogels. During thedevelopment of embodiments of the technology, experiments were conductedto test the physical characteristics (e.g., permeability to molecules,cells, etc.; stiffness) of PEG hydrogels as a function of PEGconcentration (% w/v) and/or extent and type of cross-linking. Forexample, the technology encompasses PEG hydrogels formed with PEGconcentrations of 2% to 15% (e.g., 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%,5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%,12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or 15% PEG). Furthermore, the extentof cross-linking can be controlled by controlling the exposure to asource of ultraviolet light (e.g., exposing the PEG precursor solutionfor 0.5 to 10 minutes, e.g., 0.5 minute, 1 minute, 1.5 minutes, 2minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8minutes, 8.5 minutes, 9 minutes, 9.5 minutes, or 10 minutes.

According to embodiments of the technology, controlling the PEGconcentration provides control of the intrinsic characteristics of thehydrogel, e.g., stiffness, network density, and size exclusion barrier.For example, increasing the PEG concentration (w/v) produces a stiffergel, a tighter network, and a lower exclusion barrier. Further,according to embodiments of the technology, controlling the extent ofcross-linking (e.g., by controlling the cross-linking time) providescontrol of the intrinsic characteristics of the hydrogel, e.g.,stiffness, network density, and size exclusion barrier. For example,increasing the cross-linking (e.g., by increasing the cross-linkingtime) produces a stiffer gel, tighter network, smaller pores, and lowerexclusion barrier.

Some embodiments comprise use of a non-degradable PEG, e.g.,PEG-maleimide (PEG-Mal). Some embodiments comprise use of ahydrolytically-degradable PEG, e.g., PEG-acrylate (PEG-Ac).

Anti-Inflammatory, Immunosuppressant, and Anti-Proliferative Drugs

In some embodiments, compositions according to the technology furthercomprise a drug (e.g., an anti-inflammatory drug, immunosuppressantdrug, or an anti-proliferative drug). Drugs suitable for use in thedisclosed compositions are described and can be identified usingdisclosed methods. Representative drugs include glucocorticoids,phenolic antioxidants, anti-proliferative drugs, or combinationsthereof. These are collectively referred to herein as “anti-inflammatorydrugs” unless stated otherwise.

Non-limiting examples of drugs that find use in embodiments of thedisclosed technology include steroidal anti-inflammatories such asdexamethasone, 5-FU, daunomycin, and mitomycin. Anti-angiogenic oranti-proliferative drugs are also useful.

Examples include curcumins (e.g., including monoesters andtetrahydrocurcumin) and drugs such as sirolimus (rapamycin),ciclosporin, tacrolimus, doxorubicin, mycophenolic acid, and paclitaxeland derivatives thereof. In some embodiments, the drug is an mTORinhibitor (e.g., sirolimus and everolimus) or biolimus A9, a highlylipophilic, semisynthetic sirolimus analogue with an alkoxy-alkyl groupreplacing hydrogen at position 42-O. Lisofylline is a synthetic smallmolecule with anti-inflammatory properties.

In some embodiments, the drug is a calcineurin inhibitor (e.g.,cyclosporine, pimecrolimus, and tacrolimus).

In some embodiments, the drug is a synthetic or naturalanti-inflammatory protein. In some embodiments, the device comprises anantibodies specific to select immune components. In some embodiments,the drug is an anti-T cell antibody (e.g., anti-thymocyte globulin oranti-lymphocyte globulin), anti-IL-2R alpha receptor antibody (e.g.,basiliximab or daclizumab), or anti-CD20 antibody (e.g., rituximab).

In some embodiments, the devices comprise an immunosuppressant drug, forexample, a glucocorticoid, cytostatic, antibody, drug acting onimmunophilin, and/or other immunosuppressant drug. In some embodiments,the immunosuppressant inhibits the expression and/or activity of acytokine (e.g., interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6,IL-8, and TNF-alpha) and/or the expression and/or activity of a cytokinereceptor.

Cells

The technology provides a device to encapsulate cells to protect themfrom the immune system of a transplant host and allow exchange ofnon-immune substances (e.g., molecules) between the cells and the host.Thus, the technology provides a device to immunoisolate cells from ahost immune system. While particular embodiments described herein relatein particular to the encapsulation and immunoisolation of ovary cells,the technology is not limited with respect to the cells that areincorporated into the immunoisolation device described herein. Inembodiments comprising use of ovarian cells, the encapsulated ovariancells (e.g., mammalian ovarian follicular cells) and/or ovarianfollicles are capable of producing and secreting progesterone and17ß-estradiol analogously to that which occurs in vivo.

Thus, depending on the disorder to be treated, the immunoisolationdevice can encapsulate any suitable biological material, e.g., cells,tissues, organs or parts of organs, or other biological systems or partsof biological systems. In some embodiments, the biological material isany material that is a capable of being encapsulated within the devicesdescribed (e.g., within a membrane pouch or hydrogel). For example, inexemplary embodiments the biological material is cellular material,e.g., cells or groups of cells such as acini, follicles, islets, and thelike. Typically, the biological material is a cell or group of cells ortissue that can provide a subject with some therapeutic result whenintroduced into the subject, for example the release of a bioactiveagent. The type of cell(s) will vary depending on the disorder to betreated, as is evident to those skilled in the art. For example, in thetreatment of liver failure, hepatocytes are used, and in the treatmentof diabetes, pancreatic cells (of one or more types) are used. The cellsmay be of the same type or a different type than the native tissue atthe site of implantation. For example, in the treatment of aneurological disorder in a human, human neuronal cells may beadministered, or non-human cells, such as PC12 cells derived from rats,may be administered.

Cells from a variety of sources can be used, including but not limitedto autografts (host stem-cell derived; host tissue (e.g., ovariantissue) removed and preserved (e.g., cryopreserved) prior to treatments(e.g., cytotoxic treatments), allografts (either primary cells orstem-cell derived), xenografts (porcine cells or others), or geneticallyengineered cells. In some embodiments, human biological material, orbiological material derived from humans, finds use for treatment of ahuman. In some embodiments, biological material from other sources, forexample cows, pigs, rats, sharks and sheep, finds use. Such removal maybe carried out while the donor is alive or from a dead donor. Inembodiments wherein the organs, tissues, or cells are the ovaries,ovarian tissue, or ovarian cells, the ovaries, ovarian tissue, orovarian cells are appropriately removed and washed in a physiologicalsolution.

Embodiments provide that the biological material is obtained from anysuitable source, for example research laboratories, localslaughterhouses, cell cultures, donor tissue, and the like. The numberof cells is readily controlled by means known to the skilledpractitioner. For example, in some embodiments the density of a cellsuspension is varied during formation of immunoisolation device toprovide immunoisolation devices with varied numbers of cellsencapsulated within.

The cells may be pluripotent, multipotent, totipotent, or differentiatedembryonic or adult stem cells; primary differentiated cells; orimmortalized cells, among other cell types. In certain embodiments, stemcells comprise, e.g., cells derived from cord blood, amniotic fluid,menstrual blood, placenta, Wharton's jelly, cytotropoblasts, and thelike. The cells may also comprise any combination of the above-listedcell types.

In some embodiments, the biological material comprises pancreaticislets, hepatocytes, choroid plexuses, neurons, parathyroid cells, andcells secreting clotting factors. In embodiments for the treatment ofdiabetes, the cellular material is pancreatic beta cells, pancreaticislets (Islets of Langerhans), or other insulin-producing islets capableof treating a patient suffering from diabetes. In some embodiments forthe treatment of a pancreatic exocrine disorder, the cellular materialis centroacinar cells, pancreatic basophilic cells, or acini. Inembodiments for the treatment of a pituitary disorder, the cellularmaterial is a cell of the anterior pituitary gland.

In some embodiments, the bioactive agent is any agent that is or can bereleased or secreted from the biological material. For example,pancreatic islets have the capability of secreting the bioactive agentinsulin; choroid plexuses have the capability of secreting cerebralfluids; neurons have the capability of secreting agents such as dopaminethat can affect the nervous system; and parathyroid cells have thecapability of secreting agents that can effect metabolism of calcium andphosphorus in a subject. In some embodiments, the bioactive agent is ahormone or neurotransmitter. In some embodiments for treatment of apancreatic disorder, the bioactive agent is selected from the groupconsisting of gastrin, glucagon, insulin, pancreatic polypeptide, andsomatostatin. In some embodiments for treatment of a pancreaticdisorder, the bioactive agent is selected from the group consisting ofchymotrypsin, pancreatic amylase, pancreatic lipase, and trypsin. Inembodiments for treatment of a thyroid disorder, the bioactive agent isselected from T1, T2, T3, T4, and calcitonin. In some embodiments fortreatment of a neurological disorder, the bioactive agent is aneurotransmitter, and preferably is dopamine.

In some embodiments, the cells comprise ovarian follicles. Follicles arethe functional units of the ovary, capable of secreting large amounts ofestrogen, androstenedione (the precursor of progesterone), andprogesterone. These hormones are synthesized in the somatic cells of thefollicle, theca and granulosa, after stimulation with gonadotropinssecreted from the pituitary gland in the brain. According to embodimentsof the technology, isolated ovarian follicles are encapsulated in animmunoisolation device that supported the volumetric expansion of thegrowing follicle while maintaining its spherical shape. The culture ofthe follicles contained all the required nutrients and physiologicallevels of FSH. The encapsulated follicles expanded during 8 days ofculture, which correlated with the increased levels of the secretedhormones.

In some embodiments, somatic cells (e.g., within the follicle) and/orgametes are isolated from the tissues by aspiration, centrifugation ofthe follicular liquids, or digestion of the intracellular matrix. Insome embodiments, following centrifugation, the cellular sediment iswashed by repeated passages in culture medium and recovered by removalof the supernatant. Methods are available to quantify the cellularconcentration of the sediment, e.g., as determined by direct countingusing a Makler chamber, a Bürker chamber, by cytofluorimetry, or byusing semi-automated and automated cell counters.

In some embodiments, the isolated organs, tissues, or cells aresuspended in culture or maintenance media until their encapsulation,preserving them in an environment at a temperature, in some embodiments,between ambient room temperature and −200° C. and, in some embodiments,at a humidity between 40% and 100%. In some embodiments, the isolatedorgans, tissues, or cells are suspended in culture or maintenance mediauntil their encapsulation, preserving them in an environment at atemperature between the normal body temperature of the host (e.g., ahuman having a temperature of approximately 37° C.) and −200° C.

In some embodiments, culture or maintenance media include aphysiological solution (isotonic saline), glucosate solution, BasalMedium Eagle (BME) and derivatives thereof, Hanks salts solution andderivatives thereof, tissue culture medium 199 (TCM 199) and derivativesthereof, phosphate buffered saline (PBS) and derivatives thereof, Krebssalts solution and derivatives thereof, Dulbecco modified Eagle's medium(DMEM) and derivatives thereof, tris-buffered medium (TBM) andderivatives thereof, Tyrode's salts solution and derivatives thereof,Modified sperm washing medium, modified human tubal fluid, ModifiedHam's F-10 medium, Upgraded B2 INRA medium, B2 INRA Menezo Medium,Upgraded B9 medium, and various other culture media as known and used bythose skilled in the art.

In some embodiments, the cells or tissue, suspended in culture medium orfollicular liquid, are optionally diluted into a culture medium. In someembodiments, the culture medium comprises a component (e.g., ahydrophilic polymer) that provides a synthetic extracellular matrix. Insome embodiments, dilution is between 1:0.05 and 1:200, e.g., between1:0.1 to 1:100.

In some embodiments, the cell type chosen for encapsulation in thedisclosed compositions is chosen to provide a desired therapeuticeffect. Embodiments provide that the cells may be from the patient(autologous cells), from another donor of the same species (allogeneiccells), from another species (xenogeneic), or mixtures thereof. In someembodiments, the technology comprises use of anti-inflammatory and/orimmunomodulatory (e.g., immunosuppressive) drugs to reduce the immuneresponse, e.g., provoked by the presence of the foreign hydrogelmaterials or due to the trauma of the transplant surgery. Cells can beobtained from biopsy or excision of the patient or a donor, cellculture, or cadavers. In some embodiments, cells are obtained from aculture.

In some embodiments, cells are stem cells, e.g., induced pluripotentstem cells from the subject, mobilized stem cells, mesenchymal stemcells (MSCs), etc.

In some embodiments, cells are reproductive cells, endocrine cells(e.g., ovarian cells), nervous system cells, growth factor-secretingcells, bone marrow cells, epithelial cells, endothelial cells, and/orgenetically engineered cells, among other cell types.

In some embodiments, the cells secrete a therapeutically effectivesubstance, such as a protein, nucleic acid, or small molecule (e.g., ahormone). In some embodiments, the cells metabolize toxic substances. Insome embodiments, the cells form structural tissues, such as skin, bone,cartilage, blood vessels, or muscle. In some embodiments, the cells arenatural, such as ovarian cells that naturally secrete hormones. In someembodiments, the cells are genetically engineered to express aheterologous protein or nucleic acid and/or to overexpress an endogenousprotein or nucleic acid.

Bioactive Agents

In some embodiments, bioactive agents delivered to a host by cells ofthe immunoisolation device include, e.g., insulin, glucagon,erythropoietin; Factor VIII; Factor IX; hemoglobin; albumin;neurotransmitters such as dopamine, gamma-aminobutyric acid (GABA),glutamic acid, serotonin, norepinephrine, epinephrine, andacetylcholine; growth factors such as nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin 4/5 (NT-4/5), ciliary neurotrophic factor (CNTF), glialcell line-derived neurotrophic factor (GDNF), cholinergicdifferentiation factor/leukemia inhibitory factor (CDF/LIF), epidermalgrowth factor (EGF), insulin-like growth factor (IGF), fibroblast growthfactor (FGF), and platelet-derived growth factor (PDGF); pain inhibitorssuch as Substance P, catecholamines, dynorphins, endorphins, orenkephalins; hormones such as sex hormones (e.g., estrogen,progesterone), parathyroid hormone, or growth hormone; immunomodulatorssuch as granulocyte-macrophage colony stimulating factor (GM-CSF);neuromodulators; lymphokines; cytokines; cofactors; antibodies;aptamers; and enzymes.

Mouse Model of Infertility

Embodiments of the technology were tested using a mouse model ofinfertility. In this model, both ovaries are surgically removed in ayoung female mouse (4-16 weeks old). As a result, estrogen productiondecreases and FSH levels increase because of the lack of the negativefeedback of estrogen on pituitary gland. To test embodiments of theencapsulation device, the same female receives an ovarian transplant andthe endocrine function is monitored for a predetermined time period.During the development of embodiments of the technology, encapsulatedovarian cells were tested in this model.

Kits

Some embodiments provide kits for the preparation of immunoisolatedcells in dual layer PEG devices according to the technology. Forexample, in some embodiments kits comprise previously prepared,pre-measured, and pre-packaged raw materials (e.g., degradable PEG vinylsulfone hydrogel precursor solution; non-degradable PEG vinyl sulfonehydrogel precursor solution; optionally, a buffer (e.g., HEPES buffer);a photoinitiator; and/or cross-linker (e.g., a peptide, e.g., a plasminsensitive cross-linker peptide)). Some embodiments of kits furthercomprise an ultraviolet light source as well as appropriate disposable,sterile, non-sterile, and/or sterilizable materials. The preparation ofthe encapsulation devices and/or encapsulation devices comprising cellsis performed by placing cells, tissues, tissue parts, organs, organparts, cell cores, gametes, and/or embryos into the encapsulation deviceaccording to the technology provided herein. The cells, tissues, tissueparts, organs, organ parts, cell cores, gametes, and/or embryos may befreshly removed and/or appropriately preserved according to thetechniques known to those skilled in the art.

Uses

While not limited in the uses and applications of the technologyprovided herein, the technology finds use in some embodiments to treat apatient who is in need of a bioactive substance that is produced and/orsecreted by cells, e.g., cells encapsulated in the immunoisolationdevices described herein.

The various embodiments have a wide variety of therapeutic andprophylactic uses in the area of cell therapy or cellulartransplantation, for example in the treatment of age-related disorders,allergic disorders, autoimmune diseases, cancers, endocrine disorders,immune disorders, inflammatory disorders, neurological disorders, organfailure, proliferative disorders, other conditions involving tissueinjury, and other conditions wherein replacement cells are desirable.

As known in the art, cell therapy is the transplantation of human oranimal cells to replace or repair damaged or malfunctioning tissues,and/or cells. The types of cells that are administered correspond insome way with the organ or tissue in the patient that is failing. Forexample, in the context of a subject suffering from diabetes or relateddisorders, cell therapy treatment involves the transplantation ofinsulin-producing cells that can replicate the function of pancreaticcells and release insulin into the subject upon the advent of certainconditions, namely an elevated glucose level in the subject. In thecontext of a subject suffering from impaired endocrine function (e.g.,impaired sex hormone function, e.g., impaired ovarian function), celltherapy includes transplantation of hormone producing cells (e.g.,ovarian cells).

For example, in some embodiments, the technology finds use in treating apatient (e.g., a female patient) who has been treated for a cancer,e.g., a cancer of the reproductive system. In some embodiments, thetechnology finds use for menopause hormone therapy of adult women.

In some embodiments, the technology finds use in treating a subject whohas endured an accident or an injury, e.g., the technology finds use inwound healing. In some embodiments, the device is used for transplantinga graft such as a layer or layers of cultivated, autologous, allogenic,and/or xenogenic cells to cover an accidental or surgical wound.

In some embodiments, the technology finds use in treating a subject whois transgender or who has transitioned gender.

In some embodiments, the technology finds use in treating an autoimmunedisease.

In some embodiments, the technology finds use in treating an adversedrug reaction in a subject.

In some embodiments, the technology finds use in treating adevelopmental defect.

Exemplary endocrine disorders that are treated by various embodiments ofthe present technology include, but are not limited to: adrenaldisorders, including but not limited to adrenal insufficiencies such asAddison's disease, congenital adrenal hyperplasia (adrenogenitalsyndrome), and mineralocorticoid deficiency, Conn's syndrome, Cushing'ssyndrome, and pheochromocytoma; autoimmune polyendocrine syndromes,including but not limited to Type 1 autoimmune polyendocrine syndrome,Type 2 autoimmune polyendocrine syndrome (Schmidt's syndrome), andimmunodysregulation polyendocrinopathy enteropathy X-linked syndrome(IPEX or XPID); glucose homeostasis disorders, including but not limitedto diabetes mellitus, hypoglycemia, and idiopathic hypoglycemia;metabolic bone diseases, including but not limited to osteoporosis,osteitis deformans (Paget's disease of bone), rickets and osteomalacia;pancreatic disorders, including but not limited to diabetes mellitus,exocrine pancreatic insufficiency, hypoglycemia, pancreatitis, andShwachman-Diamond Syndrome; parathyroid gland disorders, including butnot limited to primary hyperparathyroidism, secondaryhyperparathyroidism, tertiary hyperparathyroidism, hypoparathyroidism,and pseudohypoparathyroidism; pituitary gland disorders, including butnot limited to diabetes insipidus, growth hormone deficiency,hypopituitarism (or panhypopituitarism), Sheehan syndrome, and syndromeof inappropriate antidiuretic hormone; sex hormone disorders, includingbut not limited to amenorrhea, infertility, hypogonadism, gonadotropindeficiency, Kallmann syndrome, Klinefelter syndrome, menopause,menstrual function disorders, ovarian failure, polycystic ovarysyndrome, testicular failure, and Turner syndrome; and thyroiddisorders, including but not limited to hyperthyroidism, hypothyroidism,and thyroiditis, for example acute thyroiditis, De Quervain thyroiditis,Graves-Basedow disease, Hashimoto's thyroiditis, Hashitoxicosis,iatrogenic hyperthyroidism, iatrogenic hypothyroidism, Ord'sthyroiditis, postoperative hypothyroidism, postpartum thyroiditis,silent thyroiditis, thyroid storm, toxic nodular struma (Plummer'sdisease), and toxic thyroid nodule.

Exemplary cancers (e.g., of the endocrine organs) that are treated byvarious embodiments of the present technology include, but are notlimited to: adrenal hyperplasia or neoplasia, adrenocortical carcinoma,insulinoma, pituitary tumors such as pituitary adenomas, prolactinoma(or hyperprolactinemia), acromegaly (gigantism), and Cushing's disease,thyroid tumors such as thyroid adenoma, anaplastic thyroid cancer,follicular thyroid cancer, medullary thyroid cancer, and papillarythyroid cancer, and endocrine tumor syndromes such as Carney Complex,McCune-Albright syndrome, von Hippel Lindau syndrome (VHL syndrome), andmultiple endocrine neoplasia (multiple endocrine adenomatosis) or MENsyndromes such as Wermer syndrome (MEN 1), Sipple syndrome (MEN 2A), MEN2B, and FMTC.

It is contemplated that the technology finds use to implant cells thatproduce endogenous hormones or other factors at physiological levels inresponse to a stimulator. For example, in some embodiments thetechnology finds use in treating Diabetes Type I and II by deliveringislets that produce and secrete insulin in response to glucose levels inthe blood. In some embodiments, the technology finds use in deliveringcells that produce and secrete growth hormone to treat disordersaffecting the pituitary gland and production of growth hormone. In someembodiments, induced pluripotent cells derived from the patient (e.g.,autologous cells) or donor stem cells (e.g., allogeneic cells) aredifferentiated into particular cell types (e.g., ovarian cells) anddelivered to a host (e.g., transplanted) in the device.

In some embodiments, the technology finds use for the implantation ofthyroid or parathyroid tissue in a subject to treat conditionsassociated with hypoparathyroidism or hypothyroidism.

In some embodiments, the technology finds use for the implantation ofliver cells in a subject, e.g., to treat acute liver failure. Inexemplary embodiments, the implanted cells metabolize toxic metabolites,such as ammonia, and secrete urea, which is removed from the body.

In sum, embodiments of the technology find use to control drug and/orhormone release in a host without the negative impact of rejection orthe use of immunosuppressant drugs. That is, embodiments provide adevice and/or system that is implanted into a host to provide and/orcontrol the release of drug and/or hormone without the negative impactof rejection or the use of immunosuppressant drugs.

Subjects

In some embodiments, subjects for treatment include animals, e.g.,mammalian species such as humans, and domestic animals such as dogs,cats and the like, subject to a disease and other pathologicalconditions. A “patient” refers to a subject, preferably mammalian(including human), and preferably a large mammal.

Embodiments of the technology relate to the treatment of a biologicalorganism in need of treatment with a biological material and/or abioactive agent encapsulated in a membrane pouch or a hydrogel asdescribed herein. The technology is not limited in the subject orbiological material (or bioactive agent) provided to the subject in anembodiment of the technology described herein.

In some embodiments, the subject is a female. In some embodiments, thesubject is a juvenile or child and in some embodiments the subject is anadult (e.g., a subject who has passed the point of puberty). In someembodiments, the subject has abnormally low levels of a sex hormone. Insome embodiments, the subject has been treated for a cancer (e.g., acancer of the reproductive system and/or a cancer of another system ortissue whereupon cancer treatment has caused a decrease in the amount ofsex hormones in the subject). In some embodiments, the subject is anindividual who has low levels of a sex hormone due to natural ageingprocesses, e.g., in some embodiments, the subject is a female who isexperiencing the symptoms of menopause (e.g., a female in menopause orwho has had menopause).

Methods of Treatment

The administration of the immunoisolation device described herein may befor a “prophylactic” or “therapeutic” purpose. The administration issaid to be for a “therapeutic” purpose if the biological materialadministered is physiologically significant to provide a therapy for anactual manifestation of the disease, disorder, condition, or toalleviate a symptom that is present in the subject. In some embodiments,when provided therapeutically, the immunoisolation device is preferablyprovided at (or shortly after) the identification of a symptom, e.g., ofa disease, disorder, or condition. In some embodiments, the therapeuticadministration attenuates the severity of such disease, disorder, orcondition or to reverse the progress of such disease, disorder, orcondition, or to alleviate a symptom (that, in some embodiments, ispresent in a normal patient). The administration is said to be for a“prophylactic” purpose if the biological material administered isphysiologically significant to provide a therapy for a potentialdisease, disorder, or condition, or to provide relief from a potentialsymptom. In some embodiments, when provided prophylactically, theimmunoisolation device is provided in advance of any symptom thereof.The prophylactic administration attenuates the advance of the severityof such disease, disorder, or condition, or attenuates the severity of asymptom (that, in some embodiments, is present in a normal patient).

Providing a therapy or “treating” refers to any indicia of success inthe treatment or amelioration of an injury, pathology, or condition,including any objective or subjective parameter such as abatement,remission, diminishing of symptoms or making the injury, pathology, orcondition more tolerable to the patient, slowing in the rate ofdegeneration or decline, making the final point of degeneration lessdebilitating, or improving a patient's physical or mental well-being.The treatment or amelioration of symptoms can be based on objective orsubjective parameters, including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,the methods of the invention successfully treat a patient's endocrinedeficiency by normalizing the subject's sex hormone levels, which isevident upon administration of an assay for measuring the subject's sexhormone levels.

In some embodiments, the immunoisolation device is used as ahormone-producing system, which can be used to treat, e.g., an endocrinedisorder. For example, a pancreatic disorder can be treated byimplanting an immunoisolation device comprising one or more types ofpancreatic tissue or cells such as alpha cells, beta cells, centroacinarcells, delta cells, epsilon cells, pancreatic basophilic cells, PP cells(F cells), acini, or Islets of Langerhans into a patient. Similarly, athyroid disorder can be treated by implanting an immunoisolation devicecomprising one or more types of thyroid tissue such as thyroidepithelial cells (follicular cells), parafollicular cells, or folliclesinto a patient.

A variety of administration routes for the immunoisolation device areavailable. The particular mode selected depends upon the particularbiological material selected, whether the administration is forprevention, diagnosis, or treatment of disease, the severity of themedical disorder being treated and the desired therapeutic efficacy. Theduration of prophylactic and therapeutic treatment also varies dependingon the particular disease or condition being treated. Some diseases lendthemselves to acute treatment whereas others require long-term therapy.

Treatment using the immunoisolation device comprising a biologicalmaterial involves the transplantation of immunoisolation device into thebody cavity of the subject. This may be performed by creating a surgicalopening in the body cavity and introducing the immunoisolation deviceinto the body cavity through the opening. This may be accomplishedthrough plausibly simple techniques, such as placing the immunoisolationdevice through the opening and introducing them into the body cavity.Other techniques known in the art, such as hypodermic injections, mayalso be used.

Once inside the body cavity, some embodiments provide that theimmunoisolation device may move in the body cavity.

Generally, the immunoisolation device is surgically implanted, forexample laparoscopically, into an appropriate location in the body, andmay be placed therein or affixed to the surrounding tissue.

In an embodiment for the treatment of an endocrine disorder ordeficiency, a method of treatment comprises implantation of animmunoisolation device comprising hormone-producing cells that providesa sustained release of hormone. The device does not exhibit significantdegradation during the sustained-release period. The term “sustainedrelease,” as used herein, refers to the continual release of thebioactive agent from the biological material during instances when therelease should take place. For instance, if the biological material isan ovarian cell and the biological agent is estrogen and/orprogesterone, the ovarian cells should, after transplantation, releasethe estrogen and/or progesterone into the host any time the ovariancells recognize that the estrogen and/or progesterone level of the hosthas reached a certain point. After the estrogen and/or progesteronelevel in the host has been maintained, the ovarian cells temporarilycease or decrease secreting additional estrogen and/or progesterone.However, when the estrogen and/or progesterone levels in the host againreach a point where estrogen and/or progesterone is needed, thetemporarily-dormant ovarian cells again secrete estrogen and/orprogesterone. This type of continual release is an example of sustainedrelease.

In some embodiments, the sustained-release period is 1 to 7 days; 7 to14 days; 14 to 21 days; and, in some embodiments, 21 to 30 or more days(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more days). In someembodiments, the sustained-release period is more than 30 days, e.g., 31to 60 days, 60 to 90 days, or more. In some embodiments, thesustained-release period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 or more weeks; insome embodiments, the sustained-release period is 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 or more months.

The longer the device provides a sustained release of the bioactiveagent, the longer the patient will be functioning on the transplantedcells in the device alone without needing additional treatment.

The technology finds use in the treatment of a human, e.g., bytransplant in a human host in need of transplant of cells. For example,some embodiments provide that the device is provided by subcutaneousimplantation in the host, e.g., in the arm, abdomen, or upper back ofthe host. Such placement is minimally invasive and provides for accessto the device for retrieval of the device and/or cells and provides forreloading the device with fresh cells, if needed.

Additional aspects of the technology provided herein are describedthroughout this disclosure. Although the disclosure herein refers tocertain illustrated embodiments, it is to be understood that theseembodiments are presented by way of example and not by way oflimitation.

EXAMPLES

The technology relates to an encapsulating device, e.g., to provideimmunoisolation of a therapeutic (e.g., a drug, a tissue, a cell, etc.).Data collected from the experiments indicate that isogeneic ovariantissue encapsulated in an immunoisolating device and implanted in anovariectomized mouse restores ovarian endocrine function. The implantedovarian tissue continued to function as confirmed by histologicalanalysis of the implants and measured hormone levels in the serum. Inconclusion, ovarian tissue encapsulated in embodiments of the devicesdescribed herein responded to the physiological signals and secreted sexhormones and the levels of secreted hormones improved the hormonalprofile to normal (e.g., pre-ovariectomy) levels.

Example 1—Encapsulating Devices Comprising Two PEG Layers

In some embodiments the encapsulating device is a two-layerencapsulating device that comprises an “outer shell” and an “innercore”. In particular embodiments, the outer shell of the encapsulatingdevice is prepared with a non-degradable PEG (e.g., a PEG crosslinked byexposure to ultraviolet radiation) and the inner core is prepared with adegradable PEG (e.g., a degradable PEG hydrogel, e.g., crosslinked witha protease degradable linker peptide). The non-degradable outer shell ofthis design provides immune isolation of a therapeutic placed within theinner core and, in embodiments in which the therapeutic comprises cellsor tissues, the degradable inner core allows the implanted cell ortissue to expand as it grows. During the development of embodiments ofthe technology, embodiments of the two-layer encapsulating device wereproduced and experiments were conducted to characterize the two-layerencapsulating device in vitro. In particular, data were collecteddescribing the degree of swelling, pore size, diffusion rate, andpermeability of embodiments of the two-layer encapsulating device.Further experiments were conducted to collect data describing thebiocompatibility of the two-layer encapsulating device in an isogenicmodel and to evaluate the immunoisolation of a therapeutic by thetwo-layer encapsulating device.

Accordingly, during the development of embodiments of the technologydescribed herein, experiments were conducted to produce embodiments ofthe two-layer encapsulating device described herein. In particular, insome embodiments the two-layer encapsulating device comprised twopoly(ethylene glycol) (PEG) hydrogels, e.g., a first PEG hydrogel thatprovided a degradable inner core and a second PEG hydrogel that provideda non-degradable outer shell. Therapeutics (e.g., tissue, e.g., ovariantissue) were implanted in the inner core of embodiments of the two-layerPEG encapsulating devices and the immunoisolation provided by thedevices for the therapeutics was tested.

1.1 Materials and Methods Poly(Ethylene) Glycol (PEG)

PEG was obtained as a powder with greater than 90% purity (JenKemTechnology USA, Plano, Tex.) and used to prepare sterile solutions withvarying PEG concentrations as described below.

Degradable Hydrogel Preparation

Proteolytically degradable PEG vinyl sulfone hydrogels (“D-PEG-VS”) wereprepared with 8-arm PEG-VS (tripentaerythritol core; Mw=40 kDa) obtainedfrom JenKem, catalog number “8ARM(TP)-VS”. The 8-arm PEG-VS wasdissolved at 5% to 10% (w/v) final concentration in an isotonic HEPESbuffer (0.1 M HEPES, 0.1 M NaCl, pH 7.4) to prepare a degradable PEGvinyl sulfone hydrogel precursor solution. The degradable PEG vinylsulfone hydrogel precursor solution was then mixed with a plasminsensitive cross-linker having 3 reactive thiols at a 1:1 molar ratio of−SH and −VS groups. The plasmin sensitive cross-linker was a customsynthesis (Genscript, Piscataway, N.J.) and has the amino acid sequence:

(SEQ ID NO: 1) Ac-GCYK↓NSGCYK↓NSCG

In the amino acid sequence of the plasmin-sensitive cross-linkingpeptide, the N-terminal acetyl group is added to remove the electricalcharge on this terminal. The arrows indicate the protease cleavagesites. That is, the peptide has an amino acid sequence according to:

(SEQ ID NO: 2) GCYKNSGCYKNSCGwith an N-terminal acetyl group and is cleaved by a protease (e.g.,plasmin) between lysine and asparagine in the sequence, e.g., after thelysine at position 4 and/or after the lysine at position 10.

Furthermore, during the development of embodiments of the technology, apeptide was designed that has the amino acid sequence:

(SEQ ID NO: 3) GCRDVPMS↓MRGGDRCGYK↓NSCG

This peptide is sensitive to both plasmin and MMP proteases. In theamino acid sequence of the peptide that is both plasmin-sensitive andMMP-sensitive, the arrows indicate the protease cleavage sites. That is,embodiments comprise use of a peptide that has an amino acid sequenceaccording to:

(SEQ ID NO: 4) GCRDVPMSMRGGDRCGYKNSCGthat is cleaved by a protease (e.g., MMP) between serine and methioninein the sequence at positions 8 and 9 and/or that is cleaved by aprotease (e.g., plasmin) between the lysine and asparagine in thesequence at positions 18 and 19.

Mixing the PEG vinyl sulfone hydrogel precursor solution and the plasminsensitive cross-linker initiated a Michael-Type addition reaction thatwas allowed to proceed for at least 5 minutes to cross-link the PEGvinyl sulfone hydrogel and produce the degradable PEG vinyl sulfonehydrogel.

Non-Degradable Hydrogel Preparation

Non-degradable PEG vinyl sulfone hydrogels (“ND-PEG-VS”) were preparedwith 4-arm PEG-VS (pentaerythritol core; Mw=20 kDa) obtained fromJenKem, catalog number “A7025-1” or “4ARM-VS”. The 4-arm PEG-VS wasdissolved at 5% to 10% (w/v) final concentration in sterile Dulbecco'sphosphate buffered saline (D-PBS) (pH 7.4) containing 0.4 mg/100 μl of aphotoinitiator (e.g., such as an alpha hydroxy ketone, e.g.,2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone sold as IRGACURE2959 (BASF, Material No. 55047962) (see, e.g., Elisseeff et al. (2005)Biomaterials 26(11): 1211-18, incorporated herein by reference)) and0.1% (v/v) N-vinyl-2-pyrrilidone (PVP) (Sigma-Aldrich, St. Louis, USA)to prepare a non-degradable PEG vinyl sulfone hydrogel precursorsolution. PVP has been shown to enhance gelation without impactingcytocompatibility (see, e.g., Lin et al (2014) Acta Biomater 10(1):104-14, incorporated herein by reference).

The non-degradable PEG vinyl sulfone hydrogel precursor solutions wereprepared at final concentrations of 5% to 10% (w/v), which is equivalentto 2.5-30 mg/100 μl of PEG-VS. The non-degradable PEG vinyl sulfonehydrogel precursor solutions were exposed to ultraviolet light at aconstant intensity (1090 μW/cm² at a distance of 4 cm) for varyingradiation times (e.g., from 3-10 minutes) to prepare the non-degradablePEG vinyl sulfone hydrogel.

Dual Hydrogel Preparation

A degradable PEG vinyl sulfone hydrogel (“D-PEG-VS”) was prepared asdescribed above to provide the “inner core” of a dual PEG hydrogelpreparation. After the gelation of the inner (degradable) core wascomplete (approximately 7-10 minutes, depending on solid concentration),the crosslinked inner core was transferred to the center of a 10-μl beadof the non-degradable PEG vinyl sulfone hydrogel precursor solution asdescribed above and exposed to ultraviolet light at a constant intensity(1090 μW/cm² at a distance of 4 cm) for varying radiation times (fromapproximately 3-10 minutes) to provide the “outer shell” around the“inner core”.

Swelling Ratio Measurements

Compositions of ND-PEG-VS and D-PEG-VS (n=5) were manufactured andsoaked in D-PBS (pH=7.4) for 24 hours. The mass swelling ratio (Q_(m))was determined as the ratio of the wet mass and dry mass(Q_(m)=m_(s)/m_(d)), wherein the wet mass (m_(s)) was the mass of thehydrogels after soaking in D-PBS for 24 hours and the dry mass (m_(d))was the theoretical polymer mass.

Rheology

Storage modulus (G′) of the two components of the dual hydrogel(ND-PEG-VS and D-PEG-VS) was measured at 37° C. using a DHR-2 Rheometer(TA Instruments, New Castle, Del.) and a parallel plate geometry(diameter of 20 mm, gap of 1000 μm). The limits of viscoelasticity weredetermined via a strain sweep experiment with strain range from 0.01% to0.1% and angular frequency from 0.1 to 100 rad/s. Storage moduli werefound at a constant strain of 0.1% and angular frequency from 0.1 to 10rad/s. The gels were prepared as described and soaked in D-PBS (pH=7.4)for 24 hours prior to the experiment.

Diffusion

Compositions of ND-PEG-VS (e.g., at 5% and 10% (w/v)) were prepared andsoaked in a range of dextran solutions (1 mg/ml) prepared using dextransof varying sizes (5, 70, 150 kDa) for 24 hours at ambient roomtemperature. After soaking, gels were washed in D-PBS to remove excessdextran solution on the surface of gels. To quantify the release of thedextrans of the various sizes from the hydrogels, hydrogels that hadabsorbed dextran for 24 hours were transferred to fresh D-PBS at ambientroom temperature after 6, 12, 30, 60, 90, and 120 minutes to maintainnear sink conditions and fluorescence was quantified using a FluoroskanAscent FL (Thermo Electron Corporation, Finland) spectrophotometer.Relative fluorescence values were correlated to concentration (ng/ml)for the respective dextran solutions. These values were then compared tocontrols, where loaded gels from dextran solutions were soaked in D-PBSfor 24 hours at ambient room temperature.

Ovariectomies in Recipient Mice

Ovariectomies were performed on adult female mice (C57Bl/6JXCBA/Ca andC57Bl/6J) aged 12-16 weeks to induce infertility. The UCUCA guidelinesfor survival surgery in rodents and the UCUCA Policy on Analgesic Use inAnimals Undergoing Surgery were followed for all the procedures. Thefemales were anesthetized by isoflurane. Preemptive analgesics wereadministered before the first cut was made. Using aseptic techniques andprocedures, a midline incision was made in the abdominal wall. Theintraperitoneal space was exposed with an abdomen retractor. The ovarieswere removed and the remaining reproductive tract was gently reinsertedinto the body cavity. The muscle layer and the skin of the mouse arethen closed with absorbable sutures in two separate layers. The animalwas then placed in a clean warmed cage for recovery. Following recovery,the animal was housed in the animal facility.

Collection of Donor Ovaries

Ovaries were collected from 6-8 days old F1 hybrid (C57Bl/6JXCBA/Ca) andBalb/c female pup mice, which have a large number of primordialfollicles, and transferred to maintenance media. The ovaries were cutinto pieces and kept in the incubator until they were encapsulated fortransplantation.

Ovary Encapsulation in D-PEG-VS, ND-PEG-VS, and Dual PEG Hydrogels

The encapsulation of the ovarian tissue was performed in a sterilebiohazard cabinet on a heating stage to minimize ambient damage to thetissue. To prepare PEG-based implants, ovarian pieces were transferredfrom the maintenance media in the incubator and were laid on ahydrophobic slide using an insulin (27G) needle. Each ovarian piece wasabout 1 to 1.5 mm³ in volume. Droplets (5 μl) of the PEG precursor werepipetted on the same glass slide and ovarian pieces were transferredinto a droplet of degradable PEG vinyl sulfone hydrogel precursorsolution. To prepare D-PEG-VS, another 5 μl of a dissolved peptidecrosslinker were added to the droplet of PEG-VS and mixed. The slide wasthen covered with a top slide and allowed to form gels for 5 minutes.After the gelation was complete, the hydrogels were ready to betransplanted.

To prepare non-degradable PEG vinyl sulfone hydrogels with ovariantissue, droplets (10 μl) of PEG vinyl sulfone precursor solutions withthe initiator and PVP were pipetted on a hydrophobic glass slide. Thepieces of ovarian tissue were placed on the glass and transferred by aneedle into the droplets. The PEG-VS precursor solutions with the tissuewere exposed to ultraviolet light at a constant intensity (1090 μW/cm²at a distance of 4 cm) for varying radiation times (e.g., from 3 to 10minutes) to prepare the non-degradable PEG vinyl sulfone hydrogel.

For encapsulation of ovarian tissue pieces in dual PEG hydrogel, ovarianpieces were transferred into a droplet of degradable PEG vinyl sulfonehydrogel precursor solution and allowed to gel as described above, thenthe gelled degradable PEG vinyl sulfone hydrogel was encapsulated in anon-degradable PEG vinyl sulfone hydrogel as described above for thepreparation of dual hydrogels.

Subcutaneous Transplantation

Ovarian constructs were implanted subcutaneously in the back of mice. Asmall incision was made on the skin of the anesthetized mice and theconstruct (D-PEG-VS, ND-PEG-VS, and dual PEG) containing the ovaries wasplaced under the skin. The skin was then closed using 5/0 absorbablesutures. The mice were then placed in a clean warmed cage for recoveryand monitored post-operatively for a minimum of 7-10 days. Mice receiveanalgesics for at least 24 hours after surgery or as needed. Aftertransplantation, the mice were euthanized at different time points (7,26, 60, and 90 days).

Blood Collection for Hormone Analysis

Lateral tail vein blood was collected weekly, 1% of the total bodyweight in a 53/4″ glass Pasteur pipette. The mouse received preemptiveanalgesia and was restrained in a mouse trap to allow easy access to thetail. The tail was cleaned with an alcohol swab. Using a sharp scalpel,a cut at the distal tail was made. At the time of sacrifice, blood wascollected via cardiac puncture, maintained at 4° C. overnight, thencentrifuged for 10 minutes and the serum collected and stored at −20° C.Samples were diluted to one-fifth or one-tenth concentrations formeasuring follicle stimulating hormone (FSH) and estradiol (E2).

Histological Analysis

Hydrogels (D-PEG-VS, ND-PEG-VS, and dual-PEG) were fixed in Bouin'sfixative at 4° C. overnight and transferred to 70% ethanol at 4° C.until they were processed. Samples were embedded in paraffin, seriallysectioned at 5-μm thickness, and stained with hematoxylin and eosin.Primordial follicles were characterized by one layer of flattenedgranulosa cells around the oocyte, primary follicles were characterizedby one layer of cuboidal granulosa cells, secondary follicles werecharacterized by two or more layers of granulosa cells, and antralfollicles were characterized by the presence of an antral cavity.

1.2 Results Histology

PEG hydrogel encapsulation device comprising ovarian tissue wasimplanted in the back of ovariectomized mouse for 30, 60, and 90 days.Histological data collected indicated that growing ovarian follicleswere present at all the developmental stages. Further, histologicalimages showed multiple growing follicles and the ovarian tissue wassurrounded by the hydrogel.

D-PEG-VS Functionality—Cyclicity and Hormone Levels

In mice implanted with D-PEG-VS for a period of up to 60 days, decreasedFSH levels were observed compared to ovariectomized levels. Followingimplantation of 10% D-PEG-VS for a period of 60 days, the FSH levelsdecreased from 70 to 35 ng/ml, which indicated functional ovariantissue.

10% D-PEG-VS Functionality—Histological Analysis and FollicularProportions

After 7 days of implantation, follicular development up to the secondarystage was observed: 43% of follicles were primordial follicles; 40% offollicles were primary follicles; 17% of follicles were secondaryfollicles. After 30 days of implantation of 10% D-PEG-VS, folliculardevelopment up to the antral stage was observed. Of the total follicularpool, 13% of follicles were primordial follicles, 38% of follicles wereprimary follicles; 38% of follicles were secondary follicles: and 10% offollicles were antral follicles.

5% D-PEG-VS Functionality—Cyclicity and Hormone Levels

After implantation of 5% D-PEG-VS for a period of 30 days, the successrate in terms of restoration of estrus cycles was 100% at the end of thetime period (n=3). Mice vaginal cytology was used for correlation ofovarian endocrine functionality. Success was characterized by observingmice that had a normal estrus cycle post-implantation of 5% D-PEG-VShydrogels.

5% D-PEG-VS Functionality—Histological Analysis and FollicularProportions

After 30 days of implantation, follicular development up to the antralstage was observed: 12% of follicles were primordial follicles; 62% offollicles were primary follicles; 23% of follicles were secondaryfollicles; and 4% of follicles were antral follicles.

ND-PEG-VS Functionality

After implantation of the ND-PEG-VS construct (n=8), 65% of mice resumedcyclicity after 1 week, 100% were cycling after 4 weeks, yet 7 weekspost-transplantation only 50% of the mice retained normal estrous cycle.This level of success correlated with FSH levels, which remainedelevated at a level of 55 ng/ml at 60 days post transplantation.Following histological analysis, the encapsulated ovarian tissue wasnecrotic after 7 and 30 days of implantation. In mice that wereimplanted with the encapsulated tissue for 7 days, 80% exhibiteddecreased FSH levels compared to ovariectomized levels. However, hormonelevels would not be expected to change after a 7-day implantation ofencapsulated ovarian tissue in the hydrogel. Importantly, failure wasnot due to rejection from the host in this syngeneic model because theovarian tissue was completely encapsulated. Without being bound bytheory, it is contemplated that the rigidity and stability (e.g., beingnon-degradable) of ND-PEG-VS hinders the follicles from growing,expanding, and maintaining functionality.

Dual PEG Functionality

Of the mice implanted with ovarian tissue encapsulated within a dual PEGconstruct in the syngeneic model (n=3), two of the mice cycledpost-transplantation (a success rate of 67%). Of the mice implanted withovarian tissue encapsulated within dual PEG in the allogeneic model(n=3), one of the mice cycled post transplantation (a success rate of33%). As described herein, the dual PEG construct comprises ovariantissue completely encapsulated in a D-PEG “inner core” and the D-PEG“inner core” is surrounded by a ND-PEG “outer shell”.

Physical Characterization

The technology contemplates combining D-PEG and ND-PEG to provide anenvironment for encapsulated follicles to grow and remain viable andprotect the encapsulated follicles from an immunological response (e.g.,in an allogeneic host). Accordingly, without being bound by theory, itis contemplated that the D-PEG is less stiff than ND-PEG; in particular,it is contemplated that the more elastic D-PEG hydrogel does not applyas much compression against expanding follicles as does the ND-PEG and,in some embodiments, that the D-PEG hydrogel is degraded by the cells asthe cells grow in the core of the device.

Swelling ratio provides a convenient measurable value to characterizethe stiffness of hydrogels. In particular, hydrogels that swellsubstantially (e.g., have a high or higher swelling ratio) are softerand have a lower storage modulus and a larger pore size compared tohydrogels that swell less. As described below, the data collectedindicated that swelling ratio increased as the PEG concentrationdecreased, indicating an inverse relationship between PEG concentrationand swelling (e.g., a low PEG concentration correlated well with a lowstorage modulus and high swelling ratio).

During the development of embodiments of the technology describedherein, experiments were conducted to measure the swelling ratio ofD-PEG and ND-PEG. Swelling ratio is inversely related to stiffness—ahigher swelling ratio indicates a less stiff gel having a lower storagemodulus. Data were collected during experiments that indicated thatD-PEG is less stiff than ND-PEG as indicated by the associated swellingratios of D-PEG and ND-PEG over a broad range of PEG concentrationstested.

Furthermore, during the development of embodiments of the technologydescribed herein, experiments were conducted to measure the swellingratios of ND-PEG-VS hydrogels as a function of PEG concentration, amountof photoinitiator, and cross-linking time (see, e.g., FIG. 1). Inparticular:

-   1) ND-PEG-VS hydrogels were produced using 0.5% photoinitiator at    2.5 mg PEG/100 μl, 5 mg PEG/100 μl, and 10 mg PEG/100 μl; and with    cross-linking times of 1 minute, 3 minutes, and 5 minutes (see,    e.g., FIG. 1A);-   2) ND-PEG-VS hydrogels were produced using 0.4% photoinitiator at    2.5 mg PEG/100 μl, 5 mg PEG/100 μl, and 10 mg PEG/100 μl; and with    cross-linking times of 3 minutes and 5 minutes(see, e.g., FIG. 1B);-   3) ND-PEG-VS hydrogels were produced using 0.3% photoinitiator at    2.5 mg PEG/100 μl, 5 mg PEG/100 μl, and 10 mg PEG/100 μl; and with    cross-linking times of 3 minutes and 5 minutes (see, e.g., FIG. 1C);    and-   4) D-PEG-VS hydrogels were produced using 5 mg PEG/100 μl and 10 mg    PEG/100 μl; and with cross-linking times of 5 minutes and 10 minutes    (see, e.g., FIG. 1D).    The data collected indicated that swelling ratio increased as the    PEG concentration decreased.

Furthermore, rheological data were collected during the development ofembodiments of the technology (see, e.g., FIG. 2). In particular, therheological data further indicates that D-PEG is less stiff than ND-PEG.In ND-PEG and D-PEG sample preparations having a PEG concentration of 5%(w/v) and cross-linked for 5 minutes, the storage modulus measured forthe ND-PEG preparation was of 3536.69±189.12 Pa and the storage modulusmeasured for D-PEG was 2495.63±242.01 Pa. Additionally, it was observedthat the photoinitiator concentration had a limited effect on thecharacteristics of the gel; accordingly, 0.4% (w/v) photoinitiator wasselected due to ease of manufacturing. With respect to the effect ofincreasing the ND-PEG concentration, the rheological results supportedthe observed swelling ratios; in particular, higher PEG concentrationswere observed to produce hydrogels having greater storage moduli.Without being bound by theory, it is contemplated that increasing thePEG concentration provides an increased number of reactive sites beingavailable to form free radicals; thus, more cross-bridges form inpreparations comprising higher PEG concentrations. For example, anincrease in PEG concentration from 5% to 10% w/v (radiated for 5minutes) results in a significant increase in storage modulus G′ from3536.69±189.12 Pa at 5% PEG to 7704.50±500.78 Pa at 10% PEG.

During the development of embodiments of the technology provided herein,experiments were conducted to determine the size exclusion barrier ofthe ND-PEG-VS outer shell (see, e.g., FIG. 3). In particular,experiments measured release of dextrans having different sizes (e.g., 5kDa, 70 kDa, and 150 kDa) from hydrogels. It was contemplated that theND-PEG-VS would be permeable to smaller size particles (e.g., having asize similar to nutrients and hormones) and would not be permeable tolarger particles (e.g., having a size similar to host immune cells andantibodies). Data collected from the experiments indicated that the 5kDa particles passed through the ND-PEG-VS hydrogel and passage of the70 kDa and 150 kDa particles were inhibited by the ND-PEG-VS hydrogel(see, e.g., FIG. 3A and FIG. 3B). In particular, the concentration ofreleased 5 kDa particles increased substantially as a function of timebefore reaching equilibrium (see, e.g., FIG. 3A and FIG. 3B). Incontrast, a small amount of particles ranging from 70 kDa to 150 kDapenetrates the gel initially (e.g., at early time points) and anequilibrium is quickly reached (see, e.g., FIG. 3A and FIG. 3B).

In addition, the data collected indicated that the proportion ofparticles that are washed off and adsorbed on the surface compared tothe particles that penetrate the gel increasing substantially as thesize of the particle increases (FIG. 3C). For example, the amount of 150kDa dextran washed off 5% ND-PEG gels was approximately 350% greaterthan the amount that was released from the gel over 24 hours. Incontrast, the amount of 5 kDa dextran washed off 5% ND-PEG gels wasapproximately 27% of the amount that was released from the gels over 24hours. Thus, these results indicate that the hydrogels exclude largersized particles and are permeable to smaller sized particles. That is,the larger the size of the particle, the more that will be excluded frompenetrating the hydrogel.

Example 2—Encapsulating Devices Comprising a BilaminarPolytetrafluorethylene Membrane

During the development of some embodiments of the technology providedherein, experiments were conducted to test the immunoisolation providedby a synthetic membrane. The synthetic membrane is a bilayer comprisingan inner semipermeable membrane made of polytetrofluoroethylene (PTFE)that is laminated to an outer membrane covered by a loose polyester mesh(e.g., commercially available as THERACYTE, TheraCyte, Inc., LagunaHills, Calif.).

During the development of embodiments of the technology, experimentswere conducted to study implantation of isogenic ovarian tissue implantsencapsulated in TheraCyte® for a period of 7 days and 30 days. Theexperiments were performed in an isogeneic mouse model to minimize oreliminate rejection of implanted tissue. It was contemplated that theseexperiments would be used to identify the best encapsulation material tosupport ovarian tissue survival and function, which would be then testedin an allogeneic mouse model to study immunoisolation.

Ovarian tissue isolated from 6-8 days old female mice was dissected into8-10 pieces and physically inserted in a TheraCyte device having acapacity of 4 μl. The device comprising the tissue was implanted in thesubcutaneous space. The tissue was explanted after 7 and 30 days andhistological sections were examined for follicle counts and population.The ovarian tissue encapsulated in the device preserved healthymorphology during the implanted period, as observed from histologicalsections of the ovarian tissue. Growing ovarian follicles at all thedevelopmental stages were observed in multiple sections.

The data collected indicated a decrease in FSH levels in theovariectomized mice after implantation of ovaries encapsulated inTheraCyte (FIG. 4 7 days (lower line); 30 days (upper line). Thisdecrease in the circulating FSH levels indicates that the ovarian graftencapsulated in TheraCyte restores the ovarian endocrine function inovariectomized mice.

Histological images taken 7 days after syngeneic transplantation showedprimordial, primary, and secondary follicles. Histological images taken30 days after syngeneic transplantation showed primordial, secondary,and antral follicles. Initially, the ovarian grafts comprised mainlysmall immature follicles at the implantation day because the tissue wastaken from 6 days old females.

After 7 days of implantation, follicular development up to the secondarystage was observed—73% were primordial follicles, 14% were primaryfollicles, and 14% were secondary follicles. After 30 days ofimplantation, follicular development up to the antral stage wasobserved. Of the total follicular pool, 41% were primordial follicles,36% were primary follicles, 21% were secondary follicles, and 2% wereantral follicles. The distribution of the growing and non-growingfollicles resembled the normal physiological state. In sum, the imagesindicated the presence of healthy live follicles distributed in normalgrowth states, which indicated successful ovarian graft survival andfunction.

In addition, decreased FSH levels were observed compared toovariectomized levels. Following implantation for a period of 30 days,the FSH levels decreased and were statistically significantly lowercompared to the ovariectomized levels.

Thus, these experiments indicated that syngeneic implantation of ovariantissue encapsulated in TheraCyte® restored endocrine function inovariectomized mice. In particular, follicular development up to theantral stage was observed, and the decrease in FSH compared to theovariectomized levels indicated the ovarian tissue encapsulated inTheraCyte® functioned to restore endocrine function.

Example 3—Physical and Mechanical Characterization of PEG Hydrogels

During the development of embodiments of the technology describedherein, experiments were conducted to characterize the physical andmechanical properties of PEG hydrogels. Data collected during theexperiments indicated the conditions and ranges of conditions thatprovide control of biological aspects of the hydrogels, such as tissuevariability, size, source, metabolic rates, and turnover.

The swelling ratio (Qm) of hydrogels provides an indicator of thephysical properties of hydrogels (e.g., UV-crosslinked hydrogels), suchas stiffness and pore size. In particular, a greater Qm is associatedwith (e.g., indicates) softer (e.g., less stiff less rigid) hydrogelsand larger pore size.

During the development of embodiments of the technology provided herein,experiments were conducted to test PEG precursors as materials for usein the immunoisolation technology described herein. Data were collectedthat provided information about the swelling ratio of PEG hydrogels as afunction of the reactivity of the active end-group; and the overallfunctionality of the 2-arm PEG, 4-arm PEG, and 8-arm PEG precursormolecules (FIG. 5). In particular, three different reactive end groupswere tested: 1) PEG-acrylate (PEG-Ac); 2) PEG-vinyl sulfone (PEG-VS);and PEG-maleimide (PEG-Mal). While PEG-Ac is susceptible to hydrolyticdegradation, both PEG-VS and PEG-Mal are not susceptible to hydrolyticdegradation. The experiments indicated that all three reactive groupsformed hydrogels with the described UV crosslinking chemistry. Dataindicated that the swelling ratio for the three PEG hydrogels were inthe same range—the swelling ratio of PEG-VS had the highest value of 35,which was followed by the lower swelling ratios of PEG-Mal and PEG-Ac(FIG. 5).

The crosslinking chemistry of PEG hydrogels can be controlled to provideproteolytically degradable (D-PEG) or proteolytically non-degradable(ND-PEG) hydrogels. For example, in some embodiments a proteolyticallydegradable PEG hydrogel comprises a crosslinking peptide that issensitive and degradable by a cell-secreted protease. An exemplarychemistry that finds use in the production of a proteolyticallydegradable PEG is a Michael-type reaction (e.g., Michael addition) forthe nucleophilic addition of a carbanion or another nucleophile to anα,ß-unsaturated carbonyl compound. And, in some embodiments, anon-degradable PEG hydrogel is formed by UV light-induced crosslinking(ND-PEG).

In some embodiments, the degradable hydrogel is used to encapsulate atissue and allow expansion of the tissue; and the non-degradable gelprovides a “shell” around the degradable gel comprising the tissue toprotect the tissue from immune recognition. Embodiments provide that adegradable hydrogel comprising the tissue is encapsulated in anon-degradable gel. Accordingly, in some embodiments, the physicalproperties (e.g., swelling and stiffness) of the non-degradable anddegradable hydrogels match. Data were collected to characterize theswelling ratio (Qm) of D-PEG and ND-PEG hydrogels, e.g., to provideinformation about their physical properties (FIG. 6). The data indicatedthat the ND-PEG (e.g., 5% ND-PEG), that serves as the shell, and theD-PEG (e.g., 5% D-PEG) that provides the core, have matching Qm ofapproximately 30 (FIG. 6).

Hydrogels are visco-elastic materials. The storage modulus (G′) of a gelcorresponds with the elastic (solid) properties of a gel; and the lossmodulus (G″) of a gel corresponds with the viscous (fluid) properties ofthe gel. During the development of embodiments of the technologyprovided herein, experiments were conducted to evaluate how theconcentration of PEG precursors (5% w/v and 10% w/v), the crosslinkingchemistry (UV versus degradable linker), and the crosslinking time (3,5, and 10 minutes) affect the viscoelastic properties of the gels.

The data collected indicated that the storage moduli (G′) of 5% ND-PEGgels was not affected by the crosslinking time and reached 3000 Pa after5 or 10 minutes of UV irradiation (FIG. 7). 5% ND-PEG did not form after3 minutes. The data further indicated that the storage moduli of 10%ND-PEG gels was approximately double compared to the storage moduli of5% ND-PEG hydrogels and increased with the increase in irradiation time(FIG. 7), thus demonstrating tunability of the system. The storagemodulus of 5% D-PEG was similar to the 5% ND-PEG (FIG. 7), as waspredicted by the Qm values (FIG. 6).

During the development of embodiments of the technology provided herein,additional experiments were conducted to test the effect of theend-group chemistry (e.g., acrylate (PEG-Ac), vinyl sulfone (PEG-VS), ormaleimide (PEG-Mal)) on the measured storage moduli (G′) and loss moduli(G″) of 8-arm PEG hydrogels formed with UV irradiation (FIG. 8). Storageand loss moduli were measured using a rheometer. The hydrogels werepositioned between the two plates of the rheometer and the moduli of thehydrogels were measured as a function of the increasing frequency (FIG.8). Data shown are the moduli of the three different hydrogel conditionscollected with the same settings. PEG-Ac and PEG-VS were the softerhydrogels reaching G′ of about 4000 Pa, while PEG-Mal was two timesstiffer. All three hydrogels demonstrated comparable viscous component(e.g., loss moduli) close to 1000 Pa (FIG. 8).

During the development of embodiments of the technology provided herein,further experiments were conducted to test the stability of ND-PEGhydrogels crosslinked with UV light. In particular, data were collectedto measure the extent of degradation of UV-linked hydrogels in aqueousand basic medium, e.g., in 5 mM buffered and NaOH solutions at 37° C.for 30 days (FIG. 9). The swelling ratio of the hydrogels did not changein any of the conditions indicating that no degradation happened overthe course of the 30 days of the experiment. These data indicate thatthe hydrogels are stable in vivo and thus protect the implanted tissue.

Example 4—In Vivo Testing of Hydrogel Implants

During the development of embodiments of the technology provided herein,experiments were conducted to test the hydrogels in vivo to provide dataand information related to the use of hydrogels as immunoisolatingdevices. In particular, the experiments indicated that theencapsulation, implantation, and function of tissue in vivo are notaffected by the hydrogels. That is, the PEG hydrogel supports thesurvival and function of implanted syngeneic ovarian tissue.

During the experiments, images were acquired through a microscope anddata were collected to evaluate PEG hydrogels before and afterimplantation in a subject. FIGS. 10A, 10B, and 10C show tissue implantedin D-PEG. FIGS. 10D, 10E, and 10F show tissue implanted in ND-PEG. FIGS.10G, 10H, and 10I show tissue implanted in the dual-layer (“Dual-PEG”comprising a ND-PEG outer layer “shell” and D-PEG inner layer “core”).

After implantation, the ovarian tissue remained encapsulated in D-PEG(A) and the ovarian tissue encapsulated in D-PEG was observed in thesubcutaneous space 30 days post implantation (B). At the end of theexperiment, the D-PEG comprising the tissue was retrieved (C).

After implantation, the ovarian tissue remained encapsulated in ND-PEG(D) and remained encapsulated in ND-PEG at the time of sacrifice (E). Atthe end of the experiment, the ND-PEG comprising the tissue wasretrieved (F).

After implantation, the ovarian tissue remained encapsulated in Dual-PEG(G) and remained encapsulated in Dual-PEG at the time of sacrifice (H).At the end of the experiment, the Dual-PEG comprising the tissue wasretrieved (I).

In FIGS. 10B, 10E, and 10H, the dotted circle indicates the localizationof the hydrogel on the mice. White arrows in FIGS. 10A, 10D, and 10Gindicate encapsulated ovarian tissue. Gray arrows in FIGS. 10A and 10Gindicate the border of D-PEG and black arrows in FIGS. 10D and 10Gindicate the border of ND-PEG. Magnification is 5× in FIGS. 10A, 10D,and 10G. All PEG hydrogels were transparent after preparation, thetissue remained encapsulated during the implanted period, and minimalfibrous capsule formation was visible when the hydrogels were retrieved.Further experiments were conducted to evaluate the survival of syngeneicovarian tissue encapsulated and implanted in ovariectomized mice after7, 30, and 60 days (FIG. 11). Histological images were acquired ofovarian tissue encapsulated in D-PEG (FIGS. 11A, 11B, 11C), ND-PEG(FIGS. 11D, 11E, 11F), and dual-PEG (FIGS. 11G, 11H, 11I).

For D-PEG implants, primordial follicles (*) were observed to form in7-day implants (FIG. 11A). Secondary (***) and antral follicles (****)were observed to form in 30-day and 60-day implants respectively (FIGS.11B, 11C).

For ND-PEG implants, primary and secondary follicles were observed after7, 30, and 60 days (FIG. 11D, 11E, 11F).

For dual-PEG implants, secondary (FIG. 11G) and antral (FIG. 11H)follicles were observed in 7-day and 30-day implants, respectively.Ovarian tissue remained encapsulated in dual PEG after 60 days ofimplantation. The ovarian tissue survived and appeared normal.Magnification 10× (FIGS. 11D, 11E, 11H, 11I); magnification 20× (FIGS.11A, 11B, 11C, 11F, 11G).

Furthermore, experiments were conducted to evaluate the survival andfunction of encapsulated ovarian tissue by monitoring the frequency ofthe estrous cycle in mice that received implants. After ovariectomy, themice cease the estrous cycle, or cyclicity, and present with continuousmetestrous. Implantation of ovarian tissue that responds to circulatinghormones by secreting sex hormones results in the resumption of thecyclic estrous. Estrous was observed in mice implanted with D-PEG,ND-PEG, and Dual PEG implants in the syngeneic model and the presence ofcornified cells at least once per week was indicated continuation ofestrus cycles (FIG. 12). The data indicated that ND-PEG implantsrestored estrous the least effectively of the materials tested. Thedual-PEG performed restored estrous best.

Ovariectomy in mice results in removal of the sex hormones normallyproduced and secreted by the ovary. Estradiol is one of the hormonesproduced in the ovary. Follicle stimulating hormone (FSH) is secretedfrom the pituitary gland and stimulates the production of estradiol. Anegative feedback between FSH and estradiol maintains a coordinatedbalance between the pituitary gland and the ovarian follicles. When thelevels of estradiol decrease, the negative feedback of FSH is removedand the levels of FSH increase. Increased FSH stimulates ovaries toproduce and secrete estradiol. In the absence of ovaries, as in the caseof premature ovarian failure or after ovariectomy, increased levels ofFSH persist. During the development of embodiments of the technologydescribed herein, experiments produced data indicating that the FSHlevels are below 15 ng/mL before ovariectomy (e.g., in healthy mice) andFSH levels reach 60 ng/mL post ovariectomy (FIG. 13A, 13B). Implantationof ovarian tissue encapsulated in D-PEG results in a decrease of FSHlevels 60 days post implantation, indirectly indicating that the balancebetween the pituitary gland and the implanted ovarian tissue wasrestored (FIG. 13A). However, ND-PEG alone did not decrease the levelsof FSH after 60 days (FIG. 13B), thus indicating that degradable gelprovided around the implanted ovarian tissue promotes survival andfunction of the ovarian tissue.

Example 5—In Vivo Immunoisolation of Implanted Tissue

During the development of embodiments of the technology provided herein,experiments were conducted to evaluate hydrogels in vivo to provideimmunoisolation of implanted tissue in an allogeneic mouse model. Thedata collected indicated that the hydrogels not only support thesurvival and function of the implanted allogeneic ovarian tissue (e.g.,Example 4), but also that the PEG hydrogels protect the implanted tissuefrom inducing an immune reaction and thereby prevent rejection of theimplanted tissue.

During experiments conducted during the development of embodiments ofthe technology provided herein, data were collected to test allogeneictransfer of tissue from one mouse to another. In particular, allogeneicovarian tissue from Balb/C mice was isolated, encapsulated in D-PEG, andimplanted in ovariectomized C57BL/6 mice. Macroscopic images ofallogeneic ovarian tissue encapsulated in D-PEG and retrieved at thetime of sacrifice were acquired (FIG. 14A, 14B). The data demonstratedthat the implant remained positioned under the skin and the tissue wasencapsulated and surrounded by the hydrogel. No evidence of rejectionwas observed. In control experiments, implantation of allogeneic ovariantissue without encapsulation results in rejection and elimination of theimplanted tissue after 28 days of implantation (FIG. 15). Microscopeimages were taken at magnification of 5× (FIG. 15A) and 20× (FIG. 15B).Analysis of the images indicated destruction of the tissue and that noovarian follicles at any stage were present in the implanted tissue. Inadditional experiments that were similar to the experiments performedwith syngeneic tissue implantation, ovariectomy causes cessation of theestrous cycle in mice and the mice present with continuous metestrous.Implantation of the allogeneic ovarian tissue without theimmunoisolating device did not result in sustained cyclicity (FIG. 16A).In contract, estrous was observed in mice implanted with allogeneictissue encapsulated in D-PEG (FIG. 16B), in the commercially availableTheraCyte® device (FIG. 16C), and in Dual PEG (FIG. 16D) for 14 weekspost implantation.

In addition, flow cytometry analysis was used to evaluate the immunereaction towards the implanted allogeneic tissue without the device(control), healthy mice without the device (sham surgery), andencapsulated tissue (allogeneic ovarian tissue encapsulated in dual PEG)(FIG. 17). In all tested groups before implantation (Pre-TX), no IgM orIgG reactive to Balb/c were found in sera. Without immunoisolation therecipients developed IgG in approximately 21 days after receiving theallograft (highlighted with a gray square). Mice that underwent shamsurgery did not develop antibodies (negative control).

Most importantly, mice that received allograft encapsulated in dual PEGhydrogel presented no antibodies up to 60 days post implantation,similar to the negative controls. In FIG. 17, plots were obtained andmean fluorescence intensities (MFI) in the APC-channel were determinedwith FlowJo software. Gray squares depict the fraction of thymocytesbound to IgG. Together with the cyclicity and FSH results (above), thesedata indicate that PEG hydrogels protect the allogeneic ovarian tissuefrom rejection while maintaining the survival and function of theimplanted tissue.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the following claims.

1-21. (canceled)
 22. An immunoisolation device comprising: a) an inner core comprising a cell; and b) a non-degradable outer shell encapsulating the inner core.
 23. The immunoisolation device of claim 22, wherein said inner core is configured to expand and contract.
 24. The immunoisolation device of claim 22, wherein said device is structured to provide biological communication between said cells and the environment outside the non-degradable outer shell.
 25. The immunoisolation device of claim 22, wherein said cell is a somatic cell.
 26. The immunoisolation device of claim 22, wherein said cell is a germ cell or a gamete.
 27. The immunoisolation device of claim 22, wherein said cell is from an autologous source.
 28. The immunoisolation device of claim 22, wherein said cell is from an allogeneic or a xenogeneic source.
 29. The immunoisolation device of claim 22, wherein said cell is a differentiated stem cell or a genetically engineered cell.
 30. The immunoisolation device of claim 22, wherein said non-degradable outer shell comprises an internal surface contacting said inner core and an external surface contacting the environment outside the immunoisolation device.
 31. The immunoisolation device of claim 22, wherein said non-degradable outer shell comprises an internal surface contacting said inner core and an external surface configured to contact a host.
 32. The immunoisolation device of claim 22, wherein said non-degradable outer shell comprises an internal surface contacting said inner core and an external surface contacting a host.
 33. The immunoisolation device of claim 22, wherein said immunoisolation device is a dual-layer immunoisolation device.
 34. The immunoisolation device of claim 22, wherein said inner core comprises a polyethylene glycol and/or wherein said non-degradable outer shell comprises a polyethylene glycol.
 35. The immunoisolation device of claim 22, wherein said cell is immunoisolated.
 36. The immunoisolation device of claim 22, wherein said inner core is a degradable inner core.
 37. The immunoisolation device of claim 22, wherein said inner core further comprises a hormone.
 38. The immunoisolation device of claim 22, further comprising an anti-inflammatory drug, an immunosuppressant drug, or an anti-proliferative drug.
 39. The immunoisolation device of claim 22, wherein a tissue comprises said cell. 